Methods of using conjugated toxin peptide therapeutic agents

ABSTRACT

Disclosed is a composition of matter comprising an OSK1 peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are DNAs encoding the inventive composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

This application claims priority from U.S. Provisional Application No. 60/854,674, filed Oct. 25, 2006, and U.S. Application No. 60/995,370, filed Sep. 25, 2007, both of which are hereby incorporated by reference.

This application is related to U.S. Non-provisional Application Ser. No. 11/584,177, filed Oct. 19, 2006, which claims priority from U.S. Provisional Application No. 60/729,083, filed Oct. 21, 2005, both of which applications are hereby incorporated by reference.

The instant application contains an ASCII “txt” compliant sequence listing submitted via EFS-WEB on May 18, 2010, which serves as both the computer readable form (CRF) and the paper copy required by 37 C.F.R. Section 1.821(c) and 1.821(e), and is hereby incorporated by reference in its entirety. The name of the “txt” file created on Sep. 11, 2009, is: A-1186-US-NP4-RevSeqList091109-Submitted 102909.txt, and is 2,607 kB in size.

Throughout this application various publications are referenced within parentheses or brackets. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the biochemical arts, in particular to therapeutic peptides and conjugates.

2. Discussion of the Related Art

Ion channels are a diverse group of molecules that permit the exchange of small inorganic ions across membranes. All cells require ion channels for function, but this is especially so for excitable cells such as those present in the nervous system and the heart. The electrical signals orchestrated by ion channels control the thinking brain, the beating heart and the contracting muscle. Ion channels play a role in regulating cell volume, and they control a wide variety of signaling processes.

The ion channel family includes Na⁺, K⁺, and Ca²⁺ cation and Cl⁻ anion channels. Collectively, ion channels are distinguished as either ligand-gated or voltage-gated. Ligand-gated channels include both extracellular and intracellular ligand-gated channels. The extracellular ligand-gated channels include the nicotinic acetylcholine receptor (nAChR), the serotonin (5-hdroxytryptamine, 5-HT) receptors, the glycine and γ-butyric acid receptors (GABA) and the glutamate-activated channels including kanate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand gated channels include those activated by cyclic nucleotides (e.g. cAMP, cGMP), Ca²⁺ and G-proteins. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). The voltage-gated ion channels are categorized by their selectivity for inorganic ion species, including sodium, potassium, calcium and chloride ion channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).

A unified nomenclature for classification of voltage-gated channels was recently presented. (Catterall et al. (2000), Pharmacol. Rev. 55: 573-4; Gutman et al. (2000), Pharmacol. Rev. 55, 583-6; Catterall et al. (2000) Pharmacol. Rev. 55: 579-81; Catterall et al. (2000), Pharmacol. Rev. 55: 575-8; Hofmann et al. (2000), Pharmacol. Rev. 55: 587-9; Clapham et al. (2000), Pharmacol Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldin et al. (2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5).

The K⁺ channels constitute the largest and best characterized family of ion channels described to date. Potassium channels are subdivided into three general groups: the 6 transmembrane (6™) K⁺ channels, the 2™-2™/leak K⁺ channels and the 2™/K⁺ inward rectifying channels. (Tang et al. (2004), Ann. Rev. Physiol. 66, 131-159). These three groups are further subdivided into families based on sequence similarity. The voltage-gated K⁺ channels, including (Kv1-6, Kv8-9), EAG, KQT, and Slo (BKCa), are family members of the 6™ group. The 2™-2™ group comprises TWIK, TREK, TASK, TRAAK, and THIK, whereas the 2™/Kir group consists of Kir1-7. Two additional classes of ion channels include the inward rectifier potassium (IRK) and ATP-gated purinergic (P2X) channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).

Toxin peptides produced by a variety of organisms have evolved to target ion channels. Snakes, scorpions, spiders, bees, snails and sea anemone are a few examples of organisms that produce venom that can serve as a rich source of small bioactive toxin peptides or “toxins” that potently and selectively target ion channels and receptors. In most cases, these toxin peptides have evolved as potent antagonists or inhibitors of ion channels, by binding to the channel pore and physically blocking the ion conduction pathway. In some other cases, as with some of the tarantula toxin peptides, the peptide is found to antagonize channel function by binding to a region outside the pore (e.g., the voltage sensor domain).

The toxin peptides are usually between about 20 and about 80 amino acids in length, contain 2-5 disulfide linkages and form a very compact structure (see, e.g., FIG. 10). Toxin peptides (e.g., from the venom of scorpions, sea anemones and cone snails) have been isolated and characterized for their impact on ion channels. Such peptides appear to have evolved from a relatively small number of structural frameworks that are particularly well suited to addressing the critical issues of potency and stability. The majority of scorpion and Conus toxin peptides, for example, contain 10-40 amino acids and up to five disulfide bonds, forming extremely compact and constrained structure (microproteins) often resistant to proteolysis. The conotoxin and scorpion toxin peptides can be divided into a number of superfamilies based on their disulfide connections and peptide folds. The solution structure of many of these has been determined by NMR spectroscopy, illustrating their compact structure and verifying conservation of their family fold. (E.g., Tudor et al., Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin, Eur. J. Biochem. 251(1-2):133-41 (1998); Pennington et al., Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin, Biochem. 38(44): 14549-58 (1999); Jaravine et al., Three-dimensional structure of toxin OSK1 from Orthochirus scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997); del Rio-Portillo et al.; NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity, Eur. J. Biochem. 271(12): 2504-16 (2004); Prochnicka-Chalufour et al., Solution structure of discrepin, a new K⁺-channel blocking peptide from the alpha-KTx15 subfamily, Biochem. 45(6):1795-1804 (2006)).

Conserved disulfide structures can also reflect the individual pharmacological activity of the toxin family. (Nicke et al. (2004), Eur. J. Biochem. 271: 2305-19, Table 1; Adams (1999), Drug Develop. Res. 46: 219-34). For example, α-conotoxins have well-defined four cysteine/two disulfide loop structures (Loughnan, 2004) and inhibit nicotinic acetylcholine receptors. In contrast, ω-conotoxins have six cysteine/three disulfide loop consensus structures (Nielsen, 2000) and block calcium channels. Structural subsets of toxins have evolved to inhibit either voltage-gated or calcium-activated potassium channels. FIG. 9 shows that a limited number of conserved disulfide architectures shared by a variety of venomous animals from bee to snail and scorpion to snake target ion channels. FIG. 7A-7B shows alignment of alpha-scorpion toxin family and illustrates that a conserved structural framework is used to derive toxins targeting a vast array of potassium channels.

Due to their potent and selective blockade of specific ion channels, toxin peptides have been used for many years as tools to investigate the pharmacology of ion channels. Other than excitable cells and tissues such as those present in heart, muscle and brain, ion channels are also important to non-excitable cells such as immune cells. Accordingly, the potential therapeutic utility of toxin peptides has been considered for treating various immune disorders, in particular by inhibition of potassium channels such as Kv1.3 and IKCa1 since these channels indirectly control calcium signaling pathway in lymphocytes. [e.g., Kem et al., ShK toxin compositions and methods of use, U.S. Pat. No. 6,077,680; Lebrun et al., Neuropeptides originating in scorpion, U.S. Pat. No. 6,689,749; Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Mouhat et al., K⁺ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005); Mouhat et al., Pharmacological profiling of Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal domain, Molec. Pharmacol. 69:354-62 (2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B. S. Jensen et al. The Ca²⁺-activated K+ Channel of Intermediate Conductance: A Molecular Target for Novel Treatments?, Current Drug Targets 2:401-422 (2001); Rauer et al., Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca²⁺-activated over Voltage-gated K⁺ Channels, J. Biol. Chem. 275: 1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca²⁺-Activated Potassium Channels, Molecular Pharmacol. 63: 409-418 (2003); Chandy et al., K⁺ channels as targets for specific Immunomodulation, Trends in Pharmacol. Sciences 25: 280-289 (2004); Lewis & Garcia, Therapeutic Potential of Venom Peptides, Nat. Rev. Drug Discov. 2: 790-802 (2003)].

Small molecules inhibitors of Kv1.3 and IKCa1 potassium channels and the major calcium entry channel in T cells, CRAC, have also been developed to treat immune disorders [A. Schmitz et al. (2005) Molecul. Pharmacol. 68, 1254; K. G. Chandy et al. (2004) TIPS 25, 280; H. Wulff et al. (2001) J. Biol. Chem. 276, 32040; C. Zitt et al. (2004) J. Biol. Chem. 279, 12427], but obtaining small molecules with selectivity toward some of these targets has been difficult.

Calcium mobilization in lymphocytes is known to be a critical pathway in activation of inflammatory responses [M. W. Winslow et al. (2003) Current Opinion Immunol. 15, 299]. Compared to other cells, T cells show a unique sensitivity to increased levels of intracellular calcium and ion channels both directly and indirectly control this process. Inositol triphosphate (IP3) is the natural second messenger which activates the calcium signaling pathway. IP3 is produced following ligand-induced activation of the T cell receptor (TCR) and upon binding to its intracellular receptor (a channel) causes unloading of intracellular calcium stores. The endoplasmic reticulum provides one key calcium store. Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), also causes unloading of intracellular stores and activation of the calcium signaling pathway in lymphocytes. Therefore, thapsigargin can be used as a specific stimulus of the calcium signaling pathway in T cells. The unloading of intracellular calcium stores in T cells is known to cause activation of a calcium channel on the cell surface which allows for influx of calcium from outside the cell. This store operated calcium channel (SOCC) on T cells is referred to as “CRAC” (calcium release activated channel) and sustained influx of calcium through this channel is known to be critical for full T cell activation [S. Feske et al. (2005) J. Exp. Med. 202, 651 and N. Venkatesh et al. (2004) PNAS 101, 8969]. For many years it has been appreciated that in order to maintain continued calcium influx into T cells, the cell membrane must remain in a hyperpolarized condition through efflux of potassium ions. In T cells, potassium efflux is accomplished by the voltage-gated potassium channel Kv1.3 and the calcium-activated potassium channel IKCa1 [K. G. Chandy et al. (2004) TIPS 25, 280]. These potassium channels therefore indirectly control the calcium signaling pathway, by allowing for the necessary potassium efflux that allows for a sustained influx of calcium through CRAC.

Sustained increases in intracellular calcium activate a variety of pathways in T cells, including those leading to activation of NFAT, NF-kB and AP-1 [Quintana-A (2005) Pflugers Arch.—Eur. J. Physiol. 450, 1]. These events lead to various T cell responses including alteration of cell size and membrane organization, activation of cell surface effector molecules, cytokine production and proliferation. Several calcium sensing molecules transmit the calcium signal and orchestrate the cellular response. Calmodulin is one molecule that binds calcium, but many others have been identified (M. J. Berridge et al. (2003) Nat. Rev. Mol. Cell. Biol. 4, 517). The calcium-calmodulin dependent phosphatase calcineurin is activated upon sustained increases in intracellular calcium and dephosphorylates cytosolic NFAT. Dephosphorylated NFAT quickly translocates to the nucleus and is widely accepted as a critical transcription factor for T cell activation (F. Macian (2005) Nat. Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004) PNAS 101, 8969). Inhibitors of calcineurin, such as cyclosporin A (Neoral, Sand immune) and FK506 (Tacrolimus) are a main stay for treatment of severe immune disorders such as those resulting in rejection following solid organ transplant (I. M. Gonzalez-Pinto et al. (2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005) Transplant International 18, 140). Neoral has been approved for the treatment of transplant rejection, severe rheumatoid arthritis (D. E. Yocum et al. (2000) Rheumatol. 39, 156) and severe psoriasis (J. Koo (1998) British J. Dermatol. 139, 88). Preclinical and clinical data has also been provided suggesting calcineurin inhibitors may have utility in treatment of inflammatory bowel disease (IBD; Baumgart D C (2006) Am. J. Gastroenterol. March 30; Epub ahead of print), multiple sclerosis (Ann. Neurol. (1990) 27, 591) and asthma (S. Rohatagi et al. (2000) J. Clin. Pharmacol. 40, 1211). Lupus represents another disorder that may benefit from agents blocking activation of helper T cells. Despite the importance of calcineurin in regulating NFAT in T cells, calcineurin is also expressed in other tissues (e.g. kidney) and cyclosporine A & FK506 have a narrow safety margin due to mechanism based toxicity. Renal toxicity and hypertension are common side effects that have limited the promise of cyclosporine & FK506. Due to concerns regarding toxicity, calcineurin inhibitors are used mostly to treat only severe immune disease (Bissonnette-R et al. (2006) J. Am. Acad. Dermatol. 54, 472). Kv1.3 inhibitors offer a safer alternative to calcineurin inhibitors for the treatment of immune disorders. This is because Kv1.3 also operates to control the calcium signaling pathway in T cells, but does so through a distinct mechanism to that of calcineurin inhibitors, and evidence on Kv1.3 expression and function show that Kv1.3 has a more restricted role in T cell biology relative to calcineurin, which functions also in a variety of non-lymphoid cells and tissues.

Calcium mobilization in immune cells also activates production of the cytokines interleukin 2 (IL-2) and interferon gamma (IFNg) which are critical mediators of inflammation. IL-2 induces a variety of biological responses ranging from expansion and differentiation of CD4⁺ and CD8⁺ T cells, to enhancement of proliferation and antibody secretion by B cells, to activation of NK cells [S. L. Gaffen & K. D. Liu (2004) Cytokine 28, 109]. Secretion of IL-2 occurs quickly following T cell activation and T cells represent the predominant source of this cytokine. Shortly following activation, the high affinity IL-2 receptor (IL2-R) is upregulated on T cells endowing them with an ability to proliferate in response to IL-2. T cells, NK cells, B cells and professional antigen presenting cells (APCs) can all secrete IFNg upon activation. T cells represent the principle source of IFNg production in mediating adaptive immune responses, whereas natural killer (NK) cells & APCs are likely an important source during host defense against infection [K. Schroder et al. (2004) J. Leukoc. Biol. 75, 163]. IFNg, originally called macrophage-activating factor, upregulates antigen processing and presentation by monocytes, macrophages and dendritic cells. IFNg mediates a diverse array of biological activities in many cell types [U. Boehm et al. (1997) Annu. Rev. Immunol. 15, 749] including growth & differentiation, enhancement of NK cell activity and regulation of B cell immunoglobulin production and class switching.

CD40L is another cytokine expressed on activated T cells following calcium mobilization and upon binding to its receptor on B cells provides critical help allowing for B cell germinal center formation, B cell differentiation and antibody isotype switching. CD40L-mediated activation of CD40 on B cells can induce profound differentiation and clonal expansion of immunoglobulin (Ig) producing B cells [S. Quezada et al. (2004) Annu. Rev. Immunol. 22, 307]. The CD40 receptor can also be found on dendritic cells and CD40L signaling can mediate dendritic cell activation and differentiation as well. The antigen presenting capacity of B cells and dendritic cells is promoted by CD40L binding, further illustrating the broad role of this cytokine in adaptive immunity. Given the essential role of CD40 signaling to B cell biology, neutralizing antibodies to CD40L have been examined in preclinical and clinical studies for utility in treatment of systemic lupus erythematosis (SLE),—a disorder characterized by deposition of antibody complexes in tissues, inflammation and organ damage [J. Yazdany and J Davis (2004) Lupus 13, 377].

Production of toxin peptides is a complex process in venomous organisms, and is an even more complex process synthetically. Due to their conserved disulfide structures and need for efficient oxidative refolding, toxin peptides present challenges to synthesis. Although toxin peptides have been used for years as highly selective pharmacological inhibitors of ion channels, the high cost of synthesis and refolding of the toxin peptides and their short half-life in vivo have impeded the pursuit of these peptides as a therapeutic modality. Far more effort has been expended to identify small molecule inhibitors as therapeutic antagonists of ion channels, than has been given the toxin peptides themselves. One exception is the recent approval of the small ω-conotoxin MVIIA peptide (Ziconotide™) for treatment of intractable pain. The synthetic and refolding production process for Ziconotide™, however, is costly and only results in a small peptide product with a very short half-life in vivo (about 4 hours).

A cost-effective process for producing therapeutics, such as but not limited to, inhibitors of ion channels, is a desideratum provided by the present invention, which involves toxin peptides fused, or otherwise covalently conjugated to a vehicle.

SUMMARY OF THE INVENTION

The present invention relates to a composition of matter of the formula: (X¹)_(a)—(F¹)_(d)—(X²)_(b)—(F²)_(e)—(X³)_(c)  (I) and multimers thereof, wherein:

F¹ and F² are half-life extending moieties, and d and e are each independently 0 or 1, provided that at least one of d and e is 1;

X¹, X², and X³ are each independently -(L)_(f)-P-(L)_(g)-, and f and g are each independently 0 or 1;

P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, and at least one P is an OSK1 peptide analog;

L is an optional linker (present when f=1 and/or g=1); and

a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1.

The present invention thus concerns molecules having variations on Formula I, such as the formulae: P-(L)_(g)-F¹ (i.e., b, c, and e equal to 0);  (II) F¹-(L)_(g)-P (i.e., a, c, and e equal to 0);  (III) P-(L)_(g)-F¹-(L)_(f)-P or (X¹)_(a)—F¹—(X²)_(b) (i.e., c and e equal to 0);  (IV) F¹-(L)_(f)-P-(L)_(g)-F² (i.e., a and c equal to 0);  (V) F¹-(L)_(f)-P-(L)_(g)-F²-(L)_(f)-P (i.e., a equal to 0);  (VI) F¹—F²-(L)_(f)-P (i.e., a and b equal to 0);  (VII) P-(L)_(g)-F¹—F² (i.e., b and c equal to 0);  (VIII) P-(L)_(g)-F¹—F²-(L)_(f)-P (i.e., b equal to 0);  (IX) and any multimers of any of these, when stated conventionally with the N-terminus of the peptide(s) on the left. All of such molecules of Formulae II-IX are within the meaning of Structural Formula I. Within the meaning of Formula I, the toxin peptide (P), if more than one is present, can be independently the same or different from the OSK1 peptide analog, or any other toxin peptide(s) also present in the inventive composition, and the linker moiety ((L)_(f) and/or (L)_(g)), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. Conjugation of the toxin peptide(s) to the half-life extending moiety, or moieties, can be via the N-terminal and/or C-terminal of the toxin peptide, or can be intercalary as to its primary amino acid sequence, F¹ being linked closer to the toxin peptide's N-terminus than is linked F². Examples of useful half-life extending moieties (F¹ or F²) include immunoglobulin Fc domain (e.g., a human immunoglobulin Fc domain, including Fc of IgG1, IgG2, IgG3 or IgG4) or a portion thereof, human serum albumin (HSA), or poly(ethylene glycol) (PEG). These and other half-life extending moieties described herein are useful, either individually or in combination. A monovalent dimeric Fc-toxin peptide fusion (as represented schematically in FIG. 2B), for example, an Fc-OSK1 peptide analog fusion or Fc-ShK peptide analog fusion, is an example of the inventive composition of matter encompassed by Formula VII above.

The present invention also relates to a composition of matter, which includes, conjugated or unconjugated, a toxin peptide analog of ShK, OSK1, ChTx, or Maurotoxin modified from the native sequences at one or more amino acid residues, having greater Kv1.3 or IKCa1 antagonist activity, and/or target selectivity, compared to a ShK, OSK1, or Maurotoxin (MTX) peptides having a native sequence. The toxin peptide analogs comprise an amino acid sequence selected from any of the following:

SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884 through 949, or 1295 through 1300 as set forth in Table 2; or

SEQ ID NOS: 980 through 1274, 1303, or 1308 as set forth in Table 7; or any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Table 7A, Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G, Table 7H, Table 7I or Table 7J.

SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13; or

SEQ ID NOS: 36, 59, 344-346, or 1369 through 1390 as set forth in Table 14.

The present invention also relates to other toxin peptide analogs that comprise an amino acid sequence selected from, or comprise the amino acid primary sequence of, any of the following:

SEQ ID NOS: 201 through 225 as set forth in Table 3; or

SEQ ID NOS: 242 through 248 or 250 through 260 as set forth in Table 4; or

SEQ ID NOS: 261 through 275 as set forth in Table 5; or

SEQ ID NOS: 276 through 293 as set forth in Table 6; or

SEQ ID NOS: 299 through 315 as set forth in Table 8; or

SEQ ID NOS: 316 through 318 as set forth in Table 9; or

SEQ ID NO: 319 as set forth in Table 10; or

SEQ ID NO: 327 or 328 as set forth in Table 11; or

SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13;

SEQ ID NOS: 1369 through 1390 as set forth in Table 14; or

SEQ ID NOS: 348 through 353 as set forth in Table 16; or

SEQ ID NOS: 357 through 362, 364 through 368, 370, 372 through 385, or 387 through 398 as set forth in Table 19; or

SEQ ID NOS: 399 through 408 as set forth in Table 20; or

SEQ ID NOS: 410 through 421 as set forth in Table 22; or

SEQ ID NOS: 422, 424, 426, or 428 as set forth in Table 23; or

SEQ ID NOS: 430 through 437 as set forth in Table 24; or

SEQ ID NOS: 438 through 445 as set forth in Table 25; or

SEQ ID NOS: 447, 449, 451, 453, 455, or 457 as set forth in Table 26; or

SEQ ID NOS: 470 through 482 or 484 through 493 as set forth in Table 28; or

SEQ ID NOS: 495 through 506 as set forth in Table 29; or

SEQ ID NOS: 507 through 518 as set forth in Table 30.

The present invention is also directed to a pharmaceutical composition that includes the inventive composition of matter and a pharmaceutically acceptable carrier.

The compositions of this invention can be prepared by conventional synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins well known in the art. Compositions of this invention that have non-peptide portions can be synthesized by conventional organic chemistry reactions, in addition to conventional peptide chemistry reactions when applicable. Thus the present invention also relates to DNAs encoding the inventive compositions and expression vectors and host cells for recombinant expression.

The primary use contemplated is as therapeutic and/or prophylactic agents. The inventive compositions incorporating the toxin peptide can have activity and/or ion channel target selectivity comparable to—or even greater than—the unconjugated peptide.

Accordingly, the present invention includes a method of treating an autoimmune disorder, which involves administering to a patient who has been diagnosed with an autoimmune disorder, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease (IBD, including Crohn's Disease and ulcerative colitis), contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, a therapeutically effective amount of the inventive composition of matter (preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonist peptide), whereby at least one symptom of the disorder is alleviated in the patient. In addition, the present invention also relates to the use of one or more of the inventive compositions of matter in the manufacture of a medicament for the treatment or prevention of an autoimmune disorder, such as, but not limited to, any of the above-listed autoimmune disorders, e.g. multiple sclerosis, type 1 diabetes or IBD.

The present invention is further directed to a method of preventing or mitigating a relapse of a symptom of multiple sclerosis, which method involves administering to a patient, who has previously experienced at least one symptom of multiple sclerosis, a prophylactically effective amount of the inventive composition of matter (preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonist peptide), such that the at least one symptom of multiple sclerosis is prevented from recurring or is mitigated.

Although mostly contemplated as therapeutic agents, compositions of this invention can also be useful in screening for therapeutic or diagnostic agents. For example, one can use an Fc-peptide in an assay employing anti-Fc coated plates. The half-life extending moiety, such as Fc, can make insoluble peptides soluble and thus useful in a number of assays.

Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

U.S. Nonprovisional patent application Ser. No. 11/406,454, filed Apr. 17, 2006, is hereby incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows schematic structures of some exemplary Fc dimers that can be derived from an IgG1 antibody. “Fc” in the figure represents any of the Fc variants within the meaning of “Fc domain” herein. “X1” and “X2” represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows:

FIG. 1A and FIG. 1D: Single disulfide-bonded dimers;

FIG. 1B and FIG. 1E: Doubly disulfide-bonded dimers;

FIG. 1C and FIG. 1F: Noncovalent dimers.

FIG. 2A-C show schematic structures of some embodiments of the composition of the invention that shows a single unit of the pharmacologically active toxin peptide. FIG. 2A shows a single chain molecule and can also represent the DNA construct for the molecule. FIG. 2B shows a dimer in which the linker-peptide portion is present on only one chain of the dimer (i.e., a “monovalent” dimer). FIG. 2C shows a dimer having the peptide portion on both chains. The dimer of FIG. 2C will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in FIG. 2A. In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro.

FIG. 3A-3B shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that is optimized for mammalian expression and can be used in this invention.

FIG. 4A-4B shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 3 and 4, respectively) of human IgG1 Fc that is optimized for bacterial expression and can be used in this invention.

FIG. 5A shows the amino acid sequence of the mature ShK peptide (SEQ ID NO: 5), which can be encoded for by a nucleic acid sequence containing codons optimized for expression in mammalian cell, bacteria or yeast.

FIG. 5B shows the three disulfide bonds (—S—S—) formed by the six cysteines within the ShK peptide (SEQ ID NO: 10).

FIG. 6 shows an alignment of the voltage-gated potassium channel inhibitor Stichodactyla helianthus (ShK) with other closely related members of the sea anemone toxin family. The sequence of the 35 amino acid mature ShK toxin (Accession #P29187) isolated from the venom of Stichodactyla helianthus is shown aligned to other closely related members of the sea anemone family. The consensus sequence and predicted disulfide linkages are shown, with highly conserved residues being shaded. The HmK peptide toxin sequence shown (Swiss-Protein Accession #097436) is of the immature precursor from the Magnificent sea anemone (Radianthus magnifica; Heteractis maqnifica) and the putative signal peptide is underlined. The mature HmK peptide toxin would be predicted to be 35 amino acids in length and span residues 40 through 74. AeK is the mature peptide toxin, isolated from the venom of the sea anemone Actinia equine (Accession #P81897). The sequence of the mature peptide toxin AsKS (Accession #Q9TWG1) and BgK (Accession #P29186) isolated from the venom of the sea anemone Anemonia sulcata and Bunodosoma granulifera, respectively, are also shown. FIG. 6A shows the amino acid alignment (SEQ ID NO: 10) of ShK to other members of the sea anemone family of toxins, HmK (SEQ ID NO: 6 (Mature Peptide), (SEQ ID NO: 542, Signal and Mature Peptide portions)), AeK (SEQ ID NO: 7), AsKs (SEQ ID NO: 8), and BgK (SEQ ID NO: 9). The predicted disulfide linkages are shown and conserved residues are highlighted. (HmK, SEQ ID NO: 543; ShK, SEQ ID NO: 10; AeK, SEQ ID NO: 544; AsKS, SEQ ID NO: 545). FIG. 6B shows a disulfide linkage map for this family having 3 disulfide linkages (C1-C6, C2-C4, C3-C5).

FIG. 7A-7B shows an amino acid alignment of the alpha-scorpion toxin family of potassium channel inhibitors. (BmKK1, SEQ ID NO: 11; BmKK4, SEQ ID NO: 12; PBTx1, SEQ ID NO: 14; Tc32, SEQ ID NO: 13; BmKK6, SEQ ID NO: 15; P01, SEQ ID NO: 16; Pi2, SEQ ID NO: 17; Pi3, SEQ ID NO: 18; Pi4, SEQ ID NO: 19; MTX, SEQ ID NO: 20; Pi1, SEQ ID NO: 21; HsTx1, SEQ ID NO: 61; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; OSK1, SEQ ID NO: 25; BmKTX, SEQ ID NO: 22; HgTX1, SEQ ID NO: 27; MgTx, SEQ ID NO: 28; C11Tx1, SEQ ID NO: 29; NTX, SEQ ID NO: 30; Tc30, SEQ ID NO: 31; TsTX-Ka, SEQ ID NO: 32; PBTx3, SEQ ID NO: 33; Lqh 15-1, SEQ ID NO: 34; MartenTx, SEQ ID NO: 37; ChTx, SEQ ID NO:36; ChTx-Lq2, SEQ ID NO: 42; IbTx, SEQ ID NO: 38; SloTx, SEQ ID NO: 39; BmTx1; SEQ ID NO: 43; BuTx, SEQ ID NO: 41; AmmTx3, SEQ ID NO: 44; AaTX1, SEQ ID NO: 45; BmTX3, SEQ ID NO: 46; Tc1, SEQ ID NO: 48; OSK2, SEQ ID NO: 49; TsK, SEQ ID NO: 54; CoTx1, SEQ ID NO:55; CoTx2, SEQ ID NO: 871; BmPo5, SEQ ID NO: 60; ScyTx, SEQ ID NO: 51; P05, SEQ ID NO: 52; Tamapin, SEQ ID NO: 53; and TmTx, SEQ ID NO: 691. Highly conserved residues are shaded and a consensus sequence is listed. Subfamilies of the α-KTx are listed and are from Rodriguez de la Vega, R. C. et al. (2003) TIPS 24: 222-227. A list of some ion channels reported to antagonized is listed (IK=IKCa, BK=BKCa, SK=SKCa, Kv=voltage-gated K+ channels). Although most family members in this alignment represent the mature peptide product, several represent immature or modified forms of the peptide and these include: BmKK1, BmKK4, BmKK6, BmKTX, MartenTx, ChTx, ChTx-Lq2, BmTx1, AaTx1, BmTX3, TsK, CoTx1, BmP05.

FIG. 8 shows an alignment of toxin peptides converted to peptibodies in this invention (Apamin, SEQ ID NO: 68; HaTx1, SEQ ID NO: 494; ProTx1, SEQ ID NO: 56; PaTx2, SEQ ID NO: 57; ShK[2-35], SEQ ID NO: 92; ShK[1-35], SEQ ID NO: 5; HmK, SEQ ID NO: 6; ChTx (K32E), SEQ ID NO: 59; ChTx, SEQ ID NO: 36; IbTx, SEQ ID NO: 38; OSK1 (E16K, K20D), SEQ ID NO: 296; OSK1, SEQ ID NO: 25; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; MgTx, SEQ ID NO: 28; NTX, SEQ ID NO: 30; MTX, SEQ ID NO: 20; Pi2, SEQ ID NO: 17; HsTx1, SEQ ID NO: 61; Anuroctoxin [AnTx], SEQ ID NO: 62; BeKm1, SEQ ID NO: 63; ScyTx, SEQ ID NO: 51; ωGVIA, SEQ ID NO: 64; ωMVIIa, SEQ ID NO: 65; Ptu1, SEQ ID NO: 66; and CTX, SEQ ID NO: 67). The original sources of the toxins is indicated, as well as, the number of cysteines in each. Key ion channels targeted are listed. The alignment shows clustering of toxin peptides based on their source and ion channel target impact.

FIG. 9 shows disulfide arrangements within the toxin family. The number of disulfides and the disulfide bonding order for each subfamily is indicated. A partial list of toxins that fall within each disulfide linkage category is presented.

FIG. 10 illustrates that solution structures of toxins reveal a compact structure. Solution structures of native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula (HaTx1) indicate the 28 to 39 amino acid peptides all form a compact structure. The toxins shown have 3 or 4 disulfide linkages and fall within 4 of the 6 subfamilies shown in FIG. 9. The solution structures of native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula (HaTx1) were derived from Protein Data Bank (PDB) accession numbers 1ROO (mmdbld:5247), 1MTX (mmdbld:4064), 1TXM (mmdbld:6201), 1QUZ (mmdbld:36904), 1OMZ (mmdbld:1816) and 1D1H (mmdbld:14344) using the MMDB Entrez 3D-structure database [J. Chen et al. (2003) Nucleic Acids Res. 31, 474] and viewer.

FIG. 11A-C shows the nucleic acid (SEQ ID NO: 69 and SEQ ID NO: 1358) and encoded amino acid (SEQ ID NO:70, SEQ ID NO:1359 and SEQ ID NO: 1360) sequences of residues 5131-6660 of pAMG21ampR-Fc-pep. The sequences of the Fc domain (SEQ ID NOS: 71 and 72) exclude the five C-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the C-terminus of the Fc domain.

FIG. 11D shows a circle diagram of a peptibody bacterial expression vector pAMG21ampR-Fc-pep having a chloramphenicol acetyltransferase gene (cat; “CmR” site) that is replaced with the peptide-linker sequence.

FIG. 12A-C shows the nucleic acid (SEQ ID NO: 73 and SEQ ID NO: 1361) and encoded amino acid (SEQ ID NO:74, SEQ ID NO: 1362 and SEQ ID NO: 1363) sequences of residues 5131-6319 of pAMG21ampR-Pep-Fc. The sequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the five N-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the N-terminus of the Fc domain.

FIG. 12D shows a circle diagram of a peptibody bacterial expression vector having a zeocin resistance (ble; “ZeoR”) site that is replaced with the peptide-linker sequence.

FIG. 12E-G shows the nucleic acid (SEQ ID NO:1339) and encoded amino acid sequences of pAMG21ampR-Pep-Fc (SEQ ID NO:1340, SEQ ID NO:1341, and SEQ ID NO:1342). The sequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the five N-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the N-terminus of the Fc domain.

FIG. 13A is a circle diagram of mammalian expression vector pCDNA3.1(+) CMVi.

FIG. 13B is a circle diagram of mammalian expression vector pCDNA3.1 (+) CMVi-Fc-2×G4S-Activin RIIb that contains a Fc region from human IgG1, a 10 amino acid linker and the activin RIIb gene.

FIG. 13C is a circle diagram of the CHO expression vector pDSRa22 containing the Fc-L10-ShK[2-35] coding sequence.

FIG. 14A-14B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 77 and 78, respectively) of the molecule identified as “Fc-L10-ShK[1-35]” in Example 1 hereinafter. The L10 linker amino acid sequence (SEQ ID NO: 79) is underlined.

FIG. 15A-15B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 80 and 81, respectively) of the molecule identified as “Fc-L10-ShK[2-35]” in Example 2 hereinafter. The same L10 linker amino acid sequence (SEQ ID NO: 79) as used in Fc-L10-ShK[1-35] (FIG. 14A-14B) is underlined.

FIG. 16A-16B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 82 and 83, respectively) of the molecule identified as “Fc-L25-ShK[2-35]” in Example 2 hereinafter. The L25 linker amino acid sequence (SEQ ID NO: 84) is underlined.

FIG. 17 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) by reductive amination, which is also described in Example 32 hereinafter.

FIG. 18 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) via amide formation, which is also described in Example 34 hereinafter.

FIG. 19 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) by chemoselective oxime formation, which is also described in Example 33 hereinafter.

FIG. 20A shows a reversed-phase HPLC analysis at 214 nm and FIG. 20B shows electrospray mass analysis of folded ShK[2-35], also described as folded-“Des-Arg1-ShK” (Peptide 2).

FIG. 21 shows reversed phase HPLC analysis at 214 nm of N-terminally PEGylated ShK[2-35], also referred to as N-Terminally PEGylated-“Des-Arg1-ShK”.

FIG. 22A shows a reversed-phase HPLC analysis at 214 nm of folded ShK[1-35], also referred to as “ShK”.

FIG. 22B shows electrospray mass analysis of folded ShK[1-35], also referred to as “ShK”.

FIG. 23 illustrates a scheme for N-terminal PEGylation of ShK[2-35] (SEQ ID NO: 92 and SEQ ID NO: 58, also referred to as “Des-Arg1-ShK” or “ShK d1”) by reductive amination, which is also described in Example 31 hereinafter.

FIG. 24A shows a western blot of conditioned medium from HEK 293 cells transiently transfected with Fc-L10-ShK[1-35]. Lane 1: molecular weight markers; Lane 2: 15 μl Fc-L10-ShK; Lane 3: 10 μl Fc-L10-ShK; Lane 4: 5 μl Fc-L10-ShK; Lane 5; molecular weight markers; Lane 6: blank; Lane 7: 15 μl No DNA control; Lane 8: 10 μl No DNA control; Lane 9: 5 μl No DNA control; Lane 10; molecular weight markers.

FIG. 24B shows a western blot of with 15 μl of conditioned medium from clones of Chinese Hamster Ovary (CHO) cells stably transfected with Fc-L-ShK[1-35]. Lanes 1-15 were loaded as follows: blank, BB6, molecular weight markers, BB5, BB4, BB3, BB2, BB1, blank, BD6, BD5, molecular weight markers, BD4, BD3, BD2.

FIG. 25A shows a western blot of a non-reducing SDS-PAGE gel containing conditioned medium from 293T cells transiently transfected with Fc-L-SmIIIA.

FIG. 25B shows a western blot of a reducing SDS-PAGE gel containing conditioned medium from 293T cells transiently transfected with Fc-L-SmIIIA.

FIG. 26A shows a Spectral scan of 10 μl purified bivalent dimeric Fc-L10-ShK[1-35] product from stably transfected CHO cells diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1-cm path length quartz cuvette.

FIG. 26B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final bivalent dimeric Fc-L10-ShK[1-35] product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 26C shows size exclusion chromatography on 20 μg of the final bivalent dimeric Fc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 26D shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L10-ShK[1-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 26E shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final monovalent dimeric Fc-L10-ShK[1-35] product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards (10 μL), 0.5 μg product non-reduced (1.3 μL), blank, 2.0 μg product non-reduced (5 μL), blank, 10 μg product non-reduced (25 μL), Novex Mark12 wide range protein standards (10 μL), 0.5 μg product reduced (1.3 μL), blank, 2.0 μg product reduced (5 μL), blank, and 10 μg product reduced (25 μL).

FIG. 26F shows size exclusion chromatography on 20 μg of the final monovalent dimeric Fc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final purified bivalent dimeric Fc-L10-ShK[2-35] product from stably transfected CHO cells. Lane 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 27B shows size exclusion chromatography on 50 μg of the purified bivalent dimeric Fc-L10-ShK[2-35] injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27C shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of bivalent dimeric Fc-L5-ShK[2-35] purified from stably transfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 27D shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of bivalent dimeric Fc-L25-ShK[2-35] purified from stably transfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 27E shows a spectral scan of 10 μl of the bivalent dimeric Fc-L10-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 27F shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L110-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 27G shows a spectral scan of 10 μl of the bivalent dimeric Fc-L5-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 27H shows the size exclusion chromatography on 50 mg of the final bivalent dimeric Fc-L5-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27I shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L5-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 27J shows a Spectral scan of 20 μl of the product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 27K shows the size exclusion chromatography on 50 μg of the final bivalent dimeric Fc-L25-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 27L shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L25-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 28A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L10-KTX1 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 28B shows size exclusion chromatography on 45 μg of purified Fc-L10-KTX1 injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 28C shows a Spectral scan of 20 μl of the Fc-L10-KTX1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 28D shows a MALDI mass spectral analysis of the final sample of Fc-L10-KTX1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 29A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L-AgTx2 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 29B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L10-HaTx1 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced, spectral scan of the purified material.

FIG. 29C shows a Spectral scan of 20 μl of the Fc-L110-AgTx2 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 29D shows the Size exclusion chromatography on 20 μg of the final Fc-L10-AgTx2 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 29E shows a MALDI mass spectral analysis of the final sample of Fc-L10-AgTx2 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 29F shows a Spectral scan of 20 μl of the Fc-L10-HaTx1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 29G shows the size exclusion chromatography on 20 μg of the final Fc-L10-HaTx1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 29H shows a MALDI mass spectral analysis of the final sample of Fc-L10-HaTx1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 30A shows Fc-L10-ShK[1-35] purified from CHO cells produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing the human Kv1.3 channel.

FIG. 30B shows the time course of potassium current block by Fc-L10-ShK[1-35] at various concentrations. The IC₅₀ was estimated to be 15±2 pM (n=4 cells).

FIG. 30C shows synthetic ShK[1-35] (also referred to as “ShK” alone) produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel.

FIG. 30D shows the time course of ShK[1-35] block at various concentrations. The IC₅₀ for ShK was estimated to be 12±1 pM (n=4 cells).

FIG. 31A shows synthetic peptide analog ShK[2-35] producing a concentration dependent block of the outward potassium current as recorded from HEK293 cells stably expressing human Kv1.3 channel with an IC50 of 49±5 pM (n=3 cells).

FIG. 31B shows the CHO-derived Fc-L10-ShK[2-35] peptibody producing a concentration dependent block of the outward potassium current as recorded from HEK293 cell stably expressing human Kv1.3 channel with an IC50 of 115±18 pM (n=3 cells).

FIG. 31C shows the Fc-L5-ShK[2-35] peptibody produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel with an IC50 of 100 pM (n=3 cells).

FIG. 32A shows Fc-L-KTX1 peptibody purified from bacterial cells producing a concentration dependent block of the outward potassium current as recorded from HEK293 cell stably expressing human Kv1.3 channel.

FIG. 32B shows the time course of potassium current block by Fc-L10-KTX1 at various concentrations.

FIG. 33 shows by immunohistochemistry that CHO-derived Fc-L10-ShK[1-35] peptibody stains HEK 293 cells stably transfected with human Kv1.3 (FIG. 33A), whereas untransfected HEK 293 cells are not stained with the peptibody (FIG. 33B).

FIG. 34 shows results of an enzyme-immunoassay using fixed HEK 293 cells stably transfected with human Kv1.3. FIG. 34A shows the CHO-derived Fc-L10-ShK[1-35] (referred to here simply as “Fc-L10-ShK”) peptibody shows a dose-dependent increase in response, whereas the CHO-Fc control (“Fc control”) does not. FIG. 34B shows the Fc-L10-ShK[1-35] peptibody (referred to here as “Fc-ShK”) does not elicit a response from untransfected HEK 293 cells using similar conditions and also shows other negative controls.

FIG. 35 shows the CHO-derived Fc-L10-ShK[1-35] peptibody shows a dose-dependent inhibition of IL-2 (FIG. 35A) and IFNγ (FIG. 35B) production from PMA and αCD3 antibody stimulated human PBMCs. The peptibody shows a novel pharmacology exhibiting a complete inhibition of the response, whereas the synthetic ShK[1-35] peptide alone shows only a partial inhibition.

FIG. 36 shows the mammalian-derived Fc-L10-ShK[1-35] peptibody inhibits T cell proliferation (3H-thymidine incorporation) in human PBMCs from two normal donors stimulated with antibodies to CD3 and CD28. FIG. 36A shows the response of donor 1 and FIG. 36B the response of donor 2. Pre-incubation with the anti-CD32 (FcgRII) blocking antibody did not alter the sensitivity toward the peptibody.

FIG. 37 shows the purified CHO-derived Fc-L10-ShK[1-35] peptibody causes a dose-dependent inhibition of IL-2 (FIG. 37A) and IFNγ (FIG. 37B) production from αCD3 and αCD28 antibody stimulated human PBMCs.

FIG. 38A shows the PEGylated ShK[2-35] synthetic peptide produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel and the time course of potassium current block at various concentrations is shown in FIG. 38B.

FIG. 39A shows a spectral scan of 50 μl of the Fc-L10-ShK(1-35) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 39B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-ShK(1-35) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 39C shows the Size exclusion chromatography on 50 μg of the final Fc-L10-ShK(1-35) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 40A shows a Spectral scan of 20 μl of the Fc-L10-ShK(2-35) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 40B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-ShK(2-35) product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 40C shows the size exclusion chromatography on 50 μg of the final Fc-L10-ShK(2-35) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 40D shows a MALDI mass spectral analysis of the final sample of Fc-L10-ShK(2-35) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 41A shows spectral scan of 50 μl of the Fc-L10-OSK1 product diluted in 700 μl Formulation Buffer using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 41B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OSK1 product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 41C shows size exclusion chromatography on 123 μg of the final Fc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 41D shows liquid chromatography—mass spectral analysis of approximately 4 μg of the final Fc-L110-OSK1 sample using a Vydac C₄ column with part of the effluent directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer.

FIG. 42A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1040 and SEQ ID NO: 1041, respectively) of Fc-L10-OSK1.

FIG. 43A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1042 and SEQ ID NO: 1043, respectively) of Fc-L10-OSK1[K7S].

FIG. 44A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1044 and SEQ ID NO: 1045, respectively) of Fc-L10-OSK1[E16K,K20D].

FIG. 45A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1046 and SEQ ID NO: 1047, respectively) of Fc-L10-OSK1[K7S,E16K,K20D].

FIG. 46 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-6 were loaded as follows: 15 μl of Fc-L10-OSK1[K7S,E16K,K20D]; 15 μl of Fc-L10-OSK1[E16K,K20D]; 15 μl of Fc-L10-OSK1[K7S]; 15 μl of Fc-L10-OSK1; 15 μl of “No DNA” control; molecular weight markers.

FIG. 47 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-5 were loaded as follows: 21 of Fc-L10-OSK1; 5 μl of Fc-L10-OSK1; 10 μl of Fc-L10-OSK1; 20 ng Human IgG standard; molecular weight markers.

FIG. 48 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-13 were loaded as follows: 20 ng Human IgG standard; D1; C3; C2; B6; B5; B2; B1; A6; A5; A4; A3; A2 (5 μl of clone-conditioned medium loaded in lanes 2-13).

FIG. 49A shows a spectral scan of 50 μl of the Fc-L10-OSK1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 49B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OSK1 product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 49C shows Size exclusion chromatography on 149 μg of the final Fc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 49D shows MALDI mass spectral analysis of the final sample of Fc-L10-OsK1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 50A shows a spectral scan of 50 μl of the Fc-L10-OsK1(K7S) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 50B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(K7S) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 50C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(K7S) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 50D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1 (K7S) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 51A shows a spectral scan of 50 μl of the Fc-L10-OsK1(E16K, K20D) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 51B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(E16K, K20D) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 51C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(E16K, K20D) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 51D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1 (E16K, K20D) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 52A shows a spectral scan of 50 μl of the Fc-L110-OsK1 (K7S, E16K, K20D) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 52B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(K7S, E16K, K20D) product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 52C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(K7S, E16K, K20D) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 52D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1(K7S, E16K, K20D) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 53 shows inhibition of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by synthetic Osk1, a 38-residue toxin peptide of the Asian scorpion Orthochirus scrobiculosus venom. FIG. 53A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by the synthetic Osk1 toxin peptide. FIG. 53B shows the time course of the synthetic Osk1 toxin peptide block at various concentrations. The IC50 for the synthetic Osk1 toxin peptide was estimated to be 39±12 pM (n=4 cells).

FIG. 54 shows that modification of the synthetic OSK1 toxin peptide by fusion to the Fc-fragment of an antibody (OSK1-peptibody) retained the inhibitory activity against the human Kv1.3 channel. FIG. 54A shows a concentration dependent block of the outward potassium current recorded from HEK293 cells stably expressing human Kv1.3 channel by OSK1 linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 54B shows the time course of the Fc-L10-OSK1 block at various concentrations. The IC50 for Fc-L10-OSK1 was estimated to be 198±35 pM (n=6 cells), approximately 5-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 55 shows that a single amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel. FIG. 55A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with a single amino acid substitution (lysine to serine at the 7^(th) position from N-terminal, [K7S]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[K7S]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 55B shows the time course of potassium current block by Fc-L10-OSK1[K7S] at various concentrations. The IC50 was estimated to be 372±71 pM (n=4 cells), approximately 10-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 56 shows that a two amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel. FIG. 56A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with two amino acid substitutions (glutamic acid to lysine and lysine to aspartic acid at the 16^(th) and 20^(th) position from N-terminal respectively, [E16KK20D]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[E16KK20D]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 56B shows the time course of potassium current block by Fc-L10-OSK1[E16KK20D] at various concentrations. The IC50 was estimated to be 248±63 pM (n=3 cells), approximately 6-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 57 shows that a triple amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel, but the potency of inhibition was significantly reduced when compared to the synthetic OSK1 toxin peptide. FIG. 57A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with triple amino acid substitutions (lysine to serine, glutamic acid to lysine and lysine to aspartic acid at the 7^(th), 16^(th) and 20^(th) position from N-terminal respectively, [K7SE16KK20D]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[K7SE16KK20D]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 57B shows the time course of potassium current block by Fc-L10-OSK1[K7SE16KK20D] at various concentrations. The IC50 was estimated to be 812±84 pM (n=3 cells), approximately 21-fold less potent than the synthetic OSK1 toxin peptide.

FIG. 58 shows Standard curves for ShK (FIG. 58A) and 20K PEG-ShK[1-35] (FIG. 58B) containing linear regression equations for each Standard at a given percentage of serum.

FIG. 59 shows the pharmacokinetic profile in rats of 20K PEG ShK[1-35] molecule after IV injection.

FIG. 60 shows Kv1.3 inhibitory activity in serum samples (5%) of rats receiving a single equal molar IV injection of Kv1.3 inhibitors ShK versus 20K PEG-ShK[1-35].

FIG. 61 illustrates an Adoptive Transfer EAE model experimental design (n=5 rats per treatment group). Dosing values in microgram per kilogram (mg/kg) are based on peptide content.

FIG. 62 shows that treatment with PEG-ShK ameliorated disease in rats in the adoptive transfer EAE model. Clinical scoring: 0=No signs, 0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hind leg paralysis, 5.0=complete hind leg paralysis and incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of 5.5 to 6 died or were euthanized. Mean±sem values are shown. (n=5 rats per treatment group.)

FIG. 63 shows that treatment with PEG-ShK prevented loss of body weight in the adoptive transfer EAE model. Rats were weighed on days −1, 4, 6, and 8 (for surviving rats). Mean±sem values are shown.

FIG. 64 shows that thapsigargin-induced IL-2 production in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blocked the response. The BKCa channel inhibitor iberiotoxin (IbTx) showed no significant activity. The response of whole blood from two separate donors is shown in FIG. 64A and FIG. 64B.

FIG. 65 shows that thapsigargin-induced IFN-g production in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blocked the response. The BKCa channel inhibitor iberiotoxin (IbTx) showed no significant activity. The response of whole blood from two separate donors is shown in FIG. 65A and FIG. 65B.

FIG. 66 shows that thapsigargin-induced upregulation of CD40L on T cells in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitor cyclosporine A (CsA) also blocked the response. FIG. 66A shows results of an experiment looking at the response of total CD4+ T cells. FIG. 66B shows results of an experiment that looked at total CD4+ T cells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 66B, the BKCa channel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitor dendrotoxin-K (DTX-K) showed no significant activity.

FIG. 67 shows that thapsigargin-induced upregulation of the IL-2R on T cells in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitor cyclosporine A (CsA) also blocked the response. FIG. 67A shows results of an experiment looking at the response of total CD4+ T cells. FIG. 67B shows results of an experiment that looked at total CD4+ T cells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 67B, the BKCa channel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitor dendrotoxin-K (DTX-K) showed no significant activity.

FIG. 68 shows cation exchange chromatograms of PEG-peptide purification on SP Sepharose HP columns for PEG-Shk purification (FIG. 68A) and PEG-OSK-1 purification (FIG. 68B).

FIG. 69 shows RP-HPLC chromatograms on final PEG-peptide pools to demonstrate purity of PEG-Shk purity >99% (FIG. 69A) and PEG-Osk1 purity >97% (FIG. 69B).

FIG. 70 shows the amino acid sequence (SEQ ID NO: 976) of an exemplary FcLoop-L2-OsK1-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); OsK1 (underlined amino acid residues 142-179); and Fc C-terminal domain (amino acid residues 182-270).

FIG. 71 shows the amino acid sequence (SEQ ID NO: 977) of an exemplary FcLoop-L2-ShK-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); ShK (underlined amino acid residues 142-176); and Fc C-terminal domain (amino acid residues 179-267).

FIG. 72 shows the amino acid sequence (SEQ ID NO: 978) of an exemplary FcLoop-L2-ShK-L4 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); ShK (underlined amino acid residues 142-176); and Fc C-terminal domain (amino acid residues 181-269).

FIG. 73 shows the amino acid sequence (SEQ ID NO: 979) of an exemplary FcLoop-L4-OsK1-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); OsK1(underlined amino acid residues 144-181); and Fc C-terminal domain (amino acid residues 184-272).

FIG. 74 shows that the 20K PEGylated ShK[1-35] provided potent blockade of human Kv1.3 as determined by whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells. The data represents blockade of peak current.

FIG. 75 shows schematic structures of some other exemplary embodiments of the composition of matter of the invention. “X²” and “X³” represent toxin peptides or linker-toxin peptide combinations (i.e., -(L)_(f)-P-(L)_(g)-) as defined herein. As described herein but not shown in FIG. 75, an additional X¹ domain and one or more additional PEG moieties are also encompassed in other embodiments. The specific embodiments shown here are as follows:

FIG. 75C, FIG. 75D, FIG. 75G and FIG. 75H: show a single chain molecule and can also represent the DNA construct for the molecule.

FIG. 75A, FIG. 75B, FIG. 75E and FIG. 75F: show doubly disulfide-bonded Fc dimers (in position F²); FIG. 75A and FIG. 75B show a dimer having the toxin peptide portion on both chains in position X³; FIG. 75E and FIG. 75F show a dimer having the toxin peptide portion on both chains In position X².

FIG. 76A shows a spectral scan of 50 μl of the ShK[2-35]-Fc product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 76B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final ShK[2-35]-Fc product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 76C shows size exclusion chromatography on 70 μg of the final ShK[2-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 76D shows LC-MS analysis of the final ShK[2-35]-Fc sample using an Agilent 1100 HPCL running reverse phase chromatography, with the column effluent directly coupled to an electrospray source of a Thermo Finnigan LCQ ion trap mass spectrometer. Relevant spectra were summed and deconvoluted to mass data with the Bioworks software package.

FIG. 77A shows a spectral scan of 20 μl of the met-ShK[1-35]-Fc product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 77B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final met-ShK[1-35]-Fc product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.

FIG. 77C shows size exclusion chromatography on 93 μg of the final met-ShK[1-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 77D shows MALDI mass spectral analysis of the final met-ShK[1-35]-Fc sample analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.

FIG. 78 shows a spectral scan of 10 μl of the CH2-OSK1 fusion protein product diluted in 150 μl water (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.

FIG. 79 shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final CH2-OSK1 fusion protein product. Lane 1-7 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, and Novex Mark12 wide range protein standards.

FIG. 80 shows size exclusion chromatography on 50 μg of the final CH2-OSK1 fusion protein product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.

FIG. 81 shows liquid chromatography—mass spectral analysis of the CH2-OSK1 fusion protein sample using a Vydac C₄ column with part of the effluent directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer.

FIG. 82 shows cation exchange chromatogram of PEG-CH2-OSK1 reaction mixture. Vertical lines delineate fractions pooled to obtain mono-PEGylated CH2-OSK1.

FIG. 83 shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final PEGylated CH2-OSK1 pool. Lane 1-2 were loaded as follows: Novex Mark12 wide range protein standards, 2.0 μg product non-reduced.

FIG. 84 shows whole cell patch clamp (WCPC) and PatchXpress (PX) electrophysiology comparing the activity of OSK1[Ala-12] (SEQ ID No:1410) on human Kv1.3 and human Kv1.1 heterologously overexpressed on CHO and HEK293 cells, respectively. The table summarizes the calculated IC₅₀ values and the plots show the individual traces of the impact of various concentrations of analog on the relative Kv1.3 or Kv1.1 current (percent of control, POC).

FIG. 85 shows whole cell patch clamp electrophysiology comparing the activity of OSK1[Ala-29] (SEQ ID No:1424) on human Kv1.3 and human Kv1.1 heterologously overexpressed on CHO and HEK293 cells, respectively. Concentration response curves of OSK1[Ala-29] on CHO/Kv1.3 (circle, square and diamond, IC₅₀=0.033 nM, n=3) and on HEK/Kv1.1 (filled triangle, IC₅₀=2.7 nM, n=1).

FIG. 86 shows a dose-response curve for OSK1[Ala-29] (SEQ ID No:1424) against human Kv1.3 (CHO) (panel A) and human Kv1.1 (HEK293) (panel B) as determined by high-throughput 384-well planar patch clamp electrophysiology using the IonWorks Quattro system.

FIG. 87A-B show Western blots of Tris-glycine 4-20% SDS-PAGE (FIG. 87A with longer exposure time and FIG. 87B with shorter exposure time) of a monovalent dimeric Fc-L-ShK(2-35) molecule product expressed by and released into the conditioned media from mammalian cells transiently transfected with pTT5-Fc-Fc-L10-Shk(2-35), which was sampled after the indicated number of days. Lanes 3-8 were loaded with 20 μL of conditioned medium per lane. The immunoblot was probed with anti-human IgG-Fc-HRP (Pierce). The lanes were loaded as follows: 1) MW Markers; 2) purified Fc-L10-ShK(2-35), 10 ng; 3) 293-6E-HD (5-day); 4) 293-6E-HD (6-day); 5) 293-6E-PEI (5-day); 6) 293-6E-PEI (6-day); 7) CHO-S (5-day); 8) CHO-S (6-day). Four bands were expected in the reduced gel: Linker-Fc-Shk(2-35) (one cut at 3′ furin site; predicted MW: 33.4 kDa); Fc-ShK(2-35) (both furin sites cut; predicted MW: 30.4 kDa); Fc-linker (one cut at 5′ furin site; predicted MW: 29.1 kDa); Fc (both furin sites cut; predicted MW: 25.8 kDa). Further mass spec or amino acid sequence analysis of the individual bands is needed to identify these bands and their relative ratios.

FIG. 88 shows a western blot of serum samples from a pharmacokinetic study on monovalent dimeric Fc-ShK(1-35) in SD rats. Various times (0.083-168 hours) after a single 1 mg/kg intravenous injection of monovalent dimeric Fc-L10-ShK(1-35) (see, Example 2), blood was drawn, and serum was collected. A Costar EIA/RIA 96 well plate was coated with 2 μg/ml polyclonal goat anti-human Fc antibody overnight at 4° C. Capture antibody was removed and the plate was washed with PBST and then blocked with Blotto. After the plate was washed, serum samples diluted in PBST/0.1% BSA were added. Binding was allowed to occur at room temperature for several hours, and then the plate was again washed. Samples were eluted from the plate with reducing Laemmle buffer, heated, then run on SDS-Page gels. Run in an adjacent lane (“5 ng Control”) of the gel as a standard was 5 ng of the purified monovalent dimeric Fc-L10-ShK(1-35) fusion protein used in the pharmacokinetic study. Proteins were transferred to PVDF membranes by western blot. Membranes were blocked with Blotto followed by incubation with goat anti-Human Fc-HRP conjugated antibody. After the membranes were washed, signal was detected via chemiluminescence using a CCD camera.

FIG. 89 shows the NMR solution structure of OSK1 and sites identified by analoging to be important for Kv1.3 activity and selectivity. Space filling structures are shown in FIGS. 89A, 89B and 89D. The color rendering in FIG. 89A depicts amino acid charge. In FIG. 89B, several key OSK1 amino acid residues found to be important for Kv1.3 activity (Tables 37-40) are lightly shaded and labeled Phe25, Gly26, Lys27, Met29 and Asn30. In FIG. 89D residues Ser11, Met29 and His34 are labeled. Some analogues of these residues were found to result in improved Kv1.3 selectivity over Kv1.1 (Tables 41). FIG. 89C shows the three beta strands and single alpha helix of OSK1. The amino acid sequence of native OSK1 (SEQ ID No: 25) is shown in FIG. 89E, with residues forming the molecules beta strands (β1, β2, β3) and alpha helix (al) underlined. The OSK1 structures shown were derived from PDB:1SCO, and were rendered using Cn3D vers4.1.

FIG. 90A-D illustrates that toxin peptide inhibitors of Kv1.3 provide potent blockade of the whole blood inflammatory response. The activity of the calcineurin inhibitor cyclosporin A (FIG. 90A) and Kv1.3 peptide inhibitors ShK-Ala22 (FIG. 90B; SEQ ID No: 123), OSK1-Ala29 (FIG. 90C; SEQ ID No: 1424) and OSK1-Ala12 (FIG. 90D; SEQ ID No: 1410) were compared in the whole blood assay of inflammation (Example 46) using the same donor blood sample. The potency (IC50) of each molecule is shown, where for each panel the left curve is the impact on IL-2 production and the right curve is the impact on IFNγ production.

FIG. 91A-B shows an immunoblot analysis of expression of monovalent dimeric IgG1-Fc-L-ShK(2-35) from non-reduced SDS-PAGE. FIG. 91A shows detection of human Fc expression with goat anti-human IgG (H+L)-HRP. FIG. 91B shows detection of ShK(2-35) expression with a goat anti-mouse IgG (H+L)-HRP that cross reacts with human IgG. Lane 1: purified Fc-L10-Shk(2-35); Lane 2: conditioned medium from 293EBNA cells transiently transfected with pTT5-huIgG1+pTT5-hKappa+pCMVi-Fc-L10-ShK(2-35); Lane 3: conditioned media from 293EBNA cells transiently transfected with pTT5-huIgG2+pTT5-hKappa+pCMVi-Fc-L10-Shk(2-35); Lane 4: conditioned media from 293EBNA cells transiently transfected with pTT14 vector alone. The two arrows point to the full length huIgG (mol. wt.˜150 kDa) and monovalent dimeric huIgG-FcShK(2-35) (mol. wt.˜100 kDa); the abundant 60-kDa band is the bivalent dimeric Fc-ShK(2-35).

FIG. 92A-C shows schematic representations of an embodiment of a monovalent “hemibody”-toxin peptide fusion protein construct; the single toxin peptide is represented by an oval. FIG. 92A, which can also represent the DNA construct for the fusion protein, represents an immunoglobulin light chain (LC, open rectangle), an immunoglobulin heavy chain (HC, longer cross-hatched rectangle), and an immunoglobulin Fc domain (Fc, shorter cross-hatched rectangle), each separated by an intervening peptidyl linker sequence (thick lines) comprising at least one protease cleavage site (arrows), e.g., a furin cleavage site. FIG. 92 illustrates the association of the recombinantly expressed LC, HC, and Fc-toxin peptide components connected by the peptidyl linker sequences (thick lines) and, in FIG. 92C, the final monovalent chimeric immunoglobulin (LC+HC)-Fc (i.e., “hemibody”)-toxin peptide fusion protein after cleavage (intracellularly or extracellularly) at the protease cleavage sites, to release the linkers, and formation of disulfide bridges between the light and heavy chains and between the heavy chain and the Fc components (shown as thin horizontal lines between the LC, HC, and Fc components in FIG. 92C).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Definition of Terms

The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

“Polypeptide” and “protein” are used interchangeably herein and include a molecular chain of two or more amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” and “oligopeptides,” are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, inventive fusion proteins can be derivatized as described herein by well-known organic chemistry techniques.

The term “fusion protein” indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell as a single protein.

A “domain” of a protein is any portion of the entire protein, up to and including the complete protein, but typically comprising less than the complete protein. A domain can, but need not, fold independently of the rest of the protein chain and/or be correlated with a particular biological, biochemical, or structural function or location (e.g., a ligand binding domain, or a cytosolic, transmembrane or extracellular domain).

A “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a secretory signal peptide sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

The term “signal peptide” refers to a relatively short (3-60 amino acid residues long) peptide chain that directs the post-translational transport of a protein, e.g., its export to the extracellular space. Thus, secretory signal peptides are encompassed by “signal peptide”. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals.

The term “recombinant” indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well known molecular biological procedures. A “recombinant DNA molecule,” is comprised of segments of DNA joined together by means of such molecular biological techniques. The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and/or expresses a recombinant nucleic acid.

A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acid sequence,” as used interchangeably herein, is a polymer of nucleotides, including an oligonucleotide, a DNA, and RNA, a nucleic acid, or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence can be determined. Included are DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along the mRNA chain, and also determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the RNA sequence and for the amino acid sequence.

“Expression of a gene” or “expression of a nucleic acid” means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent post-translational modification of the polypeptide), or both transcription and translation, as indicated by the context.

The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. Genes typically include coding sequences and/or the regulatory sequences required for expression of such coding sequences. The term “gene” applies to a specific genomic or recombinant sequence, as well as to a cDNA or mRNA encoded by that sequence. A “fusion gene” contains a coding region that encodes a fusion protein. Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences including transcriptional control elements to which regulatory proteins, such as transcription factors, bind, resulting in transcription of adjacent or nearby sequences.

As used herein the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., Science 236:1237 (1987)).

The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence for the inventive recombinant fusion protein, so that the expressed fusion protein can be secreted by the recombinant host cell, for more facile isolation of the fusion protein from the cell, if desired. Such techniques are well known in the art. (E.g., Goodey, Andrew R.; et al., Peptide and DNA sequences, U.S. Pat. No. 5,302,697; Weiner et al., Compositions and methods for protein secretion, U.S. Pat. No. 6,022,952 and U.S. Pat. No. 6,335,178; Uemura et al., Protein expression vector and utilization thereof, U.S. Pat. No. 7,029,909; Ruben et al., 27 human secreted proteins, US 2003/0104400 A1).

The terms “in operable combination”, “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner or orientation that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced and/or transported.

Recombinant DNA- and/or RNA-mediated protein expression techniques, or any other methods of preparing peptides or, are applicable to the making of the inventive recombinant fusion proteins. For example, the peptides can be made in transformed host cells. Briefly, a recombinant DNA molecule, or construct, coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences encoding the peptides can be excised from DNA using suitable restriction enzymes. Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells in culture include bacteria (such as Escherichia coli sp.), yeast (such as Saccharomyces sp.) and other fungal cells, insect cells, plant cells, mammalian (including human) cells, e.g., CHO cells and HEK293 cells. Modifications can be made at the DNA level, as well. The peptide-encoding DNA sequence may be changed to codons more compatible with the chosen host cell. For E. coli, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art.

The term “half-life extending moiety” (i.e., F¹ or F² in Formula I) refers to a pharmaceutically acceptable moiety, domain, or “vehicle” covalently linked (“conjugated”) to the toxin peptide directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the toxin peptide, increases half-life or other pharmacokinetic properties such as but not limited to increasing the rate of absorption, reduces toxicity, improves solubility, increases biological activity and/or target selectivity of the toxin peptide with respect to a target ion channel of interest, increases manufacturability, and/or reduces immunogenicity of the toxin peptide, compared to an unconjugated form of the toxin peptide.

By “PEGylated peptide” is meant a peptide or protein having a polyethylene glycol (PEG) moiety covalently bound to an amino acid residue of the peptide itself or to a peptidyl or non-peptidyl linker (including but not limited to aromatic or aryl linkers) that is covalently bound to a residue of the peptide.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derivatization with coupling or activating moieties (e.g., with aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamide or a maleimide moiety). In accordance with the present invention, useful PEG includes substantially linear, straight chain PEG, branched PEG, or dendritic PEG. (See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris et al., Multiarmed, monofunctional, polymer for coupling to molecules and surfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498).

The term “peptibody” refers to molecules of Formula I in which F¹ and/or F² is an immunoglobulin Fc domain or a portion thereof, such as a CH2 domain of an Fc, or in which the toxin peptide is inserted into a human IgG1 Fc domain loop, such that F¹ and F² are each a portion of an Fc domain with a toxin peptide inserted between them (See, e.g., FIGS. 70-73 and Example 49 herein). Peptibodies of the present invention can also be PEGylated as described further herein, at either an Fc domain or portion thereof, or at the toxin peptide(s) portion of the inventive composition, or both.

The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 or IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. Several published patent documents describe exemplary Fc variants, as well as interaction with the salvage receptor. See International Applications WO 97/34 631 (published 25 Sep. 1997; WO 96/32 478, corresponding to U.S. Pat. No. 6,096,891, issued Aug. 1, 2000, hereby incorporated by reference in its entirety; and WO 04/110 472. Thus, the term “Fc variant” includes a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that can be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” includes a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. One skilled in the art can form multimers by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in FIG. 2. A “monovalent dimeric” Fc-toxin peptide fusion, or “monovalent dimer”, is a Fc-toxin peptide fusion that includes a toxin peptide conjugated with only one of the dimerized Fc domains (e.g., as represented schematically in FIG. 2B). A “bivalent dimeric” Fc-toxin peptide fusion, or “bivalent dimer”, is a Fc-toxin peptide fusion having both of the dimerized Fc domains each conjugated separately with a toxin peptide (e.g., as represented schematically in FIG. 2C).

The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴ wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of 2 to about 80 amino acid residues, with molecules of about 10 to about 60 amino acid residues preferred and those of about 30 to about 50 amino acid residues most preferred. Exemplary peptides can be randomly generated by any known method, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins. In any peptide portion of the inventive compositions, for example a toxin peptide or a peptide linker moiety described herein, additional amino acids can be included on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acid residues should not significantly interfere with the functional activity of the composition.

“Toxin peptides” include peptides having the same amino acid sequence of a naturally occurring pharmacologically active peptide that can be isolated from a venom, and also include modified peptide analogs (spelling used interchangeably with “analogues”) of such naturally occurring molecules.

The term “peptide analog” refers to a peptide having a sequence that differs from a peptide sequence existing in nature by at least one amino acid residue substitution, internal addition, or internal deletion of at least one amino acid, and/or amino- or carboxy-terminal end truncations, or additions). An “internal deletion” refers to absence of an amino acid from a sequence existing in nature at a position other than the N- or C-terminus. Likewise, an “internal addition” refers to presence of an amino acid in a sequence existing in nature at a position other than the N- or C-terminus. “Toxin peptide analogs”, such as, but not limited to, an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog, contain modifications of a native toxin peptide sequence of interest (e.g., amino acid residue substitutions, internal additions or insertions, internal deletions, and/or amino- or carboxy-terminal end truncations, or additions as previously described above) relative to a native toxin peptide sequence of interest, which is in the case of OSK1: GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (SEQ ID NO:25).

Examples of toxin peptides useful in practicing the present invention are listed in Tables 1-32. The toxin peptide (“P”, or equivalently shown as “P¹” in FIG. 2) comprises at least two intrapeptide disulfide bonds, as shown, for example, in FIG. 9. Accordingly, this invention concerns molecules comprising:

-   -   a) C¹-C³ and C²-C⁴ disulfide bonding in which C¹, C², C³, and C⁴         represent the order in which cysteine residues appear in the         primary sequence of the toxin peptide stated conventionally with         the N-terminus of the peptide on the left, with the first and         third cysteines in the amino acid sequence forming a disulfide         bond, and the second and fourth cysteines forming a disulfide         bond. Examples of toxin peptides with such a C¹-C³, C²-C⁴         disulfide bonding pattern include, but are not limited to,         apamin peptides, α-conopeptides, PnIA peptides, PnIB peptides,         and MII peptides, and analogs of any of the foregoing.     -   b) C¹-C⁶, C²-C⁴ and C³-C⁵ disulfide bonding in which, as         described above, C¹, C², C³, C⁴, C⁵ and C⁶ represent the order         of cysteine residues appearing in the primary sequence of the         toxin peptide stated conventionally with the N-terminus of the         peptide(s) on the left, with the first and sixth cysteines in         the amino acid sequence forming a disulfide bond, the second and         fourth cysteines forming a disulfide bond, and the third and         fifth cysteines forming a disulfide bond. Examples of toxin         peptides with such a C¹-C⁶, C²-C⁴, C³-C⁵ disulfide bonding         pattern include, but are not limited to, ShK, BgK, HmK, AeKS,         AsK, and DTX1, and analogs of any of the foregoing.     -   c) C¹-C⁴, C²-C⁵ and C³-C⁶ disulfide bonding in which, as         described above, C¹, C², C³, C⁴, C⁵ and C⁶ represent the order         of cysteine residues appearing in the primary sequence of the         toxin peptide stated conventionally with the N-terminus of the         peptide(s) on the left, with the first and fourth cysteines in         the amino acid sequence forming a disulfide bond, the second and         fifth cysteines forming a disulfide bond, and the third and         sixth cysteines forming a disulfide bond. Examples of toxin         peptides with such a C¹-C⁴, C²-C⁵, C³-C⁶ disulfide bonding         pattern include, but are not limited to, ChTx, MgTx, OSK1, KTX1,         AgTx2, Pi2, Pi3, NTX, HgTx1, BeKM1, BmKTX, P01, BmKK6, Tc32,         Tc1, BmTx1, BmTX3, IbTx, P05, ScyTx, TsK, HaTx1, ProTX1, PaTX2,         Ptu1, ωGVIA, ωMVIIA, and SmIIIa, and analogs of any of the         foregoing.     -   d) C¹-C⁵, C²-C⁶, C³-C⁷, and C⁴-C⁸ disulfide bonding in which C¹,         C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine         residues appearing in the primary sequence of the toxin peptide         stated conventionally with the N-terminus of the peptide(s) on         the left, with the first and fifth cysteines in the amino acid         sequence forming a disulfide bond, the second and sixth         cysteines forming a disulfide bond, the third and seventh         cysteines forming a disulfide bond, and the fourth and eighth         cysteines forming a disulfide bond. Examples of toxin peptides         with such a C¹-C⁵, C²-C⁶, C³-C⁷, C⁴-C⁸ disulfide bonding pattern         include, but are not limited to, Anuoroctoxin (AnTx), Pi1,         HsTx1, MTX (P12A, P20A), and Pi4 peptides, and analogs of any of         the foregoing.     -   e) C¹-C⁴, C²-C⁶, C³-C⁷, and C⁵-C⁸ disulfide bonding in which C⁷,         C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine         residues appearing in the primary sequence of the toxin peptide         stated conventionally with the N-terminus of the peptide(s) on         the left, with the first and fourth cysteines in the amino acid         sequence forming a disulfide bond, the second and sixth         cysteines forming a disulfide bond, the third and seventh         cysteines forming a disulfide bond, and the fifth and eighth         cysteines forming a disulfide bond. Examples of toxin peptides         with such a C¹-C⁴, C²-C⁶, C³-C⁷, C⁵-C⁸ disulfide bonding pattern         include, but are not limited to, Chlorotoxin, Bm-12b, and, and         analogs of either.     -   f) C¹-C⁵, C²-C⁶, C³-C⁴, and C⁷-C⁸ disulfide bonding in which C¹,         C², C³, C⁴, C⁵, C⁶, C⁷ and C⁸ represent the order of cysteine         residues appearing in the primary sequence of the toxin peptide         stated conventionally with the N-terminus of the peptide(s) on         the left, with the first and fifth cysteines in the amino acid         sequence forming a disulfide bond, the second and sixth         cysteines forming a disulfide bond, the third and fourth         cysteines forming a disulfide bond, and the seventh and eighth         cysteines forming a disulfide bond. Examples of toxin peptides         with such a C¹-C⁵, C²-C⁶, C³-C⁴, C⁷-C⁸ disulfide bonding pattern         include, but are not limited to, Maurotoxin peptides and analogs         thereof.

The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, yeast-based screening, RNA-peptide screening, chemical screening, rational design, protein structural analysis, and the like.

The term “pharmacologically active” means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below.

The terms “-mimetic peptide” and “-agonist peptide” refer to a peptide having biological activity comparable to a naturally occurring toxin peptide molecule, e.g., naturally occurring ShK toxin peptide. These terms further include peptides that indirectly mimic the activity of a naturally occurring toxin peptide molecule, such as by potentiating the effects of the naturally occurring molecule.

The term “antagonist peptide” or “inhibitor peptide” refers to a peptide that blocks or in some way interferes with the biological activity of a receptor of interest, or has biological activity comparable to a known antagonist or inhibitor of a receptor of interest (such as, but not limited to, an ion channel).

The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.

The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.

The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.

The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, R, N-methyl-arginine, ω-aminoarginine, ω-methyl-arginine, 1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.

The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, Q, D, E, K, and citrulline (Cit) residues.

The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).

The term “neutral polar residue” refers to amino acid residues in D- or L-form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral polar amino acid residues include A, V, L, I, P, W, M, and F.

The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N.

The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N.

In some useful embodiments of the compositions of the invention, the amino acid sequence of the toxin peptide is modified in one or more ways relative to a native toxin peptide sequence of interest, such as, but not limited to, a native ShK or OSK1 sequence, their peptide analogs, or any other toxin peptides having amino acid sequences as set for in any of Tables 1-32. The one or more useful modifications can include amino acid additions or insertions, amino acid deletions, peptide truncations, amino acid substitutions, and/or chemical derivatization of amino acid residues, accomplished by known chemical techniques. Such modifications can be, for example, for the purpose of enhanced potency, selectivity, and/or proteolytic stability, or the like. Those skilled in the art are aware of techniques for designing peptide analogs with such enhanced properties, such as alanine scanning, rational design based on alignment mediated mutagenesis using known toxin peptide sequences and/or molecular modeling. For example, ShK analogs can be designed to remove protease cleavage sites (e.g., trypsin cleavage sites at K or R residues and/or chymotrypsin cleavage sites at F, Y, or W residues) in a ShK peptide- or ShK analog-containing composition of the invention, based partially on alignment mediated mutagenesis using HmK (see, e.g., FIG. 6) and molecular modeling. (See, e.g., Kalman et al., ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707 (1998); Kem et al., U.S. Pat. No. 6,077,680; Mouhat et al., OsK1 derivatives, WO 2006/002850 A2)).

The term “protease” is synonymous with “peptidase”. Proteases comprise two groups of enzymes: the endopeptidases which cleave peptide bonds at points within the protein, and the exopeptidases, which remove one or more amino acids from either N- or C-terminus respectively. The term “proteinase” is also used as a synonym for endopeptidase. The four mechanistic classes of proteinases are: serine proteinases, cysteine proteinases, aspartic proteinases, and metallo-proteinases. In addition to these four mechanistic classes, there is a section of the enzyme nomenclature which is allocated for proteases of unidentified catalytic mechanism. This indicates that the catalytic mechanism has not been identified.

Cleavage subsite nomenclature is commonly adopted from a scheme created by Schechter and Berger (Schechter I. & Berger A., On the size of the active site in proteases. I. Papain, Biochemical and Biophysical Research Communication, 27:157 (1967); Schechter I. & Berger A., On the active site of proteases. 3. Mapping the active site of papain; specific inhibitor peptides of papain, Biochemical and Biophysical Research Communication, 32:898 (1968)). According to this model, amino acid residues in a substrate undergoing cleavage are designated P1, P2, P3, P4 etc. in the N-terminal direction from the cleaved bond. Likewise, the residues in the C-terminal direction are designated P1′, P2′, P3′, P4′. etc.

The skilled artisan is aware of a variety of tools for identifying protease binding or protease cleavage sites of interest. For example, the PeptideCutter software tool is available through the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB; expasy.org/tools/peptidecutter). PeptideCutter searches a protein sequence from the SWISS-PROT and/or TrEMBL databases or a user-entered protein sequence for protease cleavage sites. Single proteases and chemicals, a selection or the whole list of proteases and chemicals can be used. Different forms of output of the results are available: tables of cleavage sites either grouped alphabetically according to enzyme names or sequentially according to the amino acid number. A third option for output is a map of cleavage sites. The sequence and the cleavage sites mapped onto it are grouped in blocks, the size of which can be chosen by the user. Other tools are also known for determining protease cleavage sites. (E.g., Turk, B. et al., Determination of protease cleavage site motifs using mixture-based oriented peptide libraries, Nature Biotechnology, 19:661-667 (2001); Barrett A. et al., Handbook of proteolytic enzymes, Academic Press (1998)).

The serine proteinases include the chymotrypsin family, which includes mammalian protease enzymes such as chymotrypsin, trypsin or elastase or kallikrein. The serine proteinases exhibit different substrate specificities, which are related to amino acid substitutions in the various enzyme subsites interacting with the substrate residues. Some enzymes have an extended interaction site with the substrate whereas others have a specificity restricted to the P1 substrate residue.

Trypsin preferentially cleaves at R or K in position P1. A statistical study carried out by Keil (1992) described the negative influences of residues surrounding the Arg- and Lys-bonds (i.e. the positions P2 and P1′, respectively) during trypsin cleavage. (Keil, B., Specificity of proteolysis, Springer-Verlag Berlin-Heidelberg-New York, 335 (1992)). A proline residue in position P1′ normally exerts a strong negative influence on trypsin cleavage. Similarly, the positioning of R and K in P1′ results in an inhibition, as well as negatively charged residues in positions P2 and P1′.

Chymotrypsin preferentially cleaves at a W, Y or F in position P1 (high specificity) and to a lesser extent at L, M or H residue in position P1. (Keil, 1992). Exceptions to these rules are the following: When W is found in position P1, the cleavage is blocked when M or P are found in position P1′ at the same time. Furthermore, a proline residue in position P1′ nearly fully blocks the cleavage independent of the amino acids found in position P1. When an M residue is found in position P1, the cleavage is blocked by the presence of a Y residue in position P1′. Finally, when H is located in position P1, the presence of a D, M or W residue also blocks the cleavage.

Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acid residues. (Connelly, J C et al., Neutral Endopeptidase 24.11 in Human Neutrophils: Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 (1985)).

Thrombin preferentially cleaves at an R residue in position P1. (Keil, 1992). The natural substrate of thrombin is fibrinogen. Optimum cleavage sites are when an R residue is in position P1 and Gly is in position P2 and position P1′. Likewise, when hydrophobic amino acid residues are found in position P4 and position P3, a proline residue in position P2, an R residue in position P1, and non-acidic amino acid residues in position P1′ and position P2′. A very important residue for its natural substrate fibrinogen is a D residue in P10.

Caspases are a family of cysteine proteases bearing an active site with a conserved amino acid sequence and which cleave peptides specifically following D residues. (Eamshaw W C et al., Mammalian caspases: Structure, activation, substrates, and functions during apoptosis, Annual Review of Biochemistry, 68:383-424 (1999)).

The Arg-C proteinase preferentially cleaves at an R residue in position P1. The cleavage behavior seems to be only moderately affected by residues in position P1′. (Keil, 1992). The Asp-N endopeptidase cleaves specifically bonds with a D residue in position P1′. (Keil, 1992).

Furin is a ubiquitous subtilisin-like proprotein convertase. It is the major processing enzyme of the secretory pathway and intracellularly is localized in the trans-golgi network (van den Ouweland, A. M. W. et al. (1990) Nucl. Acids Res., 18, 664; Steiner, D. F. (1998) Curr. Opin. Chem. Biol., 2, 31-39). The minimal furin cleavage site is Arg-X-X-Arg′. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg′. An additional arginine at the P6 position appears to enhance cleavage (Krysan, D. J. et al. (1999) J. Biol. Chem., 274, 23229-23234).

The foregoing is merely exemplary and by no means an exhaustive treatment of knowledge available to the skilled artisan concerning protease binding and/or cleavage sites that the skilled artisan may be interested in eliminating in practicing the invention.

Additional useful embodiments of the toxin peptide, e.g., the OSK1 peptide analog, can result from conservative modifications of the amino acid sequences of the peptides disclosed herein. Conservative modifications will produce peptides having functional, physical, and chemical characteristics similar to those of the parent peptide from which such modifications are made. Such conservatively modified forms of the peptides disclosed herein are also contemplated as being an embodiment of the present invention.

In contrast, substantial modifications in the functional and/or chemical characteristics of the toxin peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the region of the substitution, for example, as an α-helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.

For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan et al., Acta Physiol. Scand. Suppl., 643:55-67 (1998); Sasaki et al., 1998, Adv. Biophys. 35:1-24 (1998), which discuss alanine scanning mutagenesis).

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-conjugated peptide molecules described herein.

Naturally occurring residues may be divided into classes based on common side chain properties:

1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.

In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine norleucine, alanine, or methionine for another, the substitution of one polar (hydrophilic) amino acid residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic amino acid residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative amino acid substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the requisite biological activity. Other exemplary amino acid substitutions that can be useful in accordance with the present invention are set forth in Table 1A.

TABLE 1A Some Useful Amino Acid Substitutions Original Exemplary Residues Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala, Phe Lys Arg, 1,4-Diamino- butyric Acid, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

In other examples, a toxin peptide amino acid sequence, e.g., an OSK1 peptide analog sequence, modified from a naturally occurring toxin peptide amino acid sequence includes at least one amino acid residue inserted or substituted therein, relative to the amino acid sequence of the native toxin peptide sequence of interest, in which the inserted or substituted amino acid residue has a side chain comprising a nucleophilic or electrophilic reactive functional group by which the peptide is conjugated to a linker or half-life extending moiety. In accordance with the invention, useful examples of such a nucleophilic or electrophilic reactive functional group include, but are not limited to, a thiol, a primary amine, a seleno, a hydrazide, an aldehyde, a carboxylic acid, a ketone, an aminooxy, a masked (protected) aldehyde, or a masked (protected) keto functional group. Examples of amino acid residues having a side chain comprising a nucleophilic reactive functional group include, but are not limited to, a lysine residue, an α,β-diaminopropionic acid residue, an α,γ-diaminobutyric acid residue, an ornithine residue, a cysteine, a homocysteine, a glutamic acid residue, an aspartic acid residue, or a selenocysteine residue. In some embodiments, the toxin peptide amino acid sequence (or “primary sequence”) is modified at one, two, three, four, five or more amino acid residue positions, by having a residue substituted therein different from the native primary sequence (e.g., OSK1 SEQ ID NO:25) or omitted (e.g., an OSK1 peptide analog optionally lacking a residue at positions 36, 37, 36-38, 37-38, or 38).

In further describing toxin peptides herein, a one-letter abbreviation system is frequently applied to designate the identities of the twenty “canonical” amino acid residues generally incorporated into naturally occurring peptides and proteins (Table 1B). Such one-letter abbreviations are entirely interchangeable in meaning with three-letter abbreviations, or non-abbreviated amino acid names. Within the one-letter abbreviation system used herein, an uppercase letter indicates a L-amino acid, and a lower case letter indicates a D-amino acid, unless otherwise noted herein. For example, the abbreviation “R” designates L-arginine and the abbreviation “r” designates D-arginine.

TABLE 1B One-letter abbreviations for the canonical amino acids Three-letter abbreviations are in parentheses Alanine (Ala) A Glutamine (Gln) Q Leucine (Leu) L Serine (Ser) S Arginine (Arg) R Glutamic Acid (Glu) E Lysine (Lys) K Threonine (Thr) T Asparagine (Asn) N Glycine (Gly) G Methionine (Met) M Tryptophan (Trp) W Aspartic Acid (Asp) D Histidine (His) H Phenylalanine (Phe) F Tyrosine (Tyr) Y Cysteine (Cys) C Isoleucine (Ile) I Proline (Pro) P Valine (Val) V

An amino acid substitution in an amino acid sequence is typically designated herein with a one-letter abbreviation for the amino acid residue in a particular position, followed by the numerical amino acid position relative to the native toxin peptide sequence of interest, which is then followed by the one-letter symbol for the amino acid residue substituted in. For example, “T30D” symbolizes a substitution of a threonine residue by an aspartate residue at amino acid position 30, relative to a hypothetical native toxin peptide sequence. By way of further example, “R18hR” or “R18Cit” indicates a substitution of an arginine residue by a homoarginine or a citrulline residue, respectively, at amino acid position 18, relative to the hypothetical native toxin peptide. An amino acid position within the amino acid sequence of any particular toxin peptide (or peptide analog) described herein may differ from its position relative to the native sequence, i.e., as determined in an alignment of the N-terminal or C-terminal end of the peptide's amino acid sequence with the N-terminal or C-terminal end, as appropriate, of the native toxin peptide sequence. For example, amino acid position 1 of the sequence SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK(2-35); SEQ ID NO:92), a N-terminal truncation of the native ShK sequence, thus aligned with the C-terminal of native ShK(1-35) (SEQ ID NO:5), corresponds to amino acid position 2 relative to the native sequence, and amino acid position 34 of SEQ ID NO:92 corresponds to amino acid position 35 relative to the native sequence (SEQ ID NO:5).

In certain embodiments of the present invention, amino acid substitutions encompass, non-canonical amino acid residues, which include naturally rare (in peptides or proteins) amino acid residues or unnatural amino acid residues. Non-canonical amino acid residues can be incorporated into the peptide by chemical peptide synthesis rather than by synthesis in biological systems, such as recombinantly expressing cells, or alternatively the skilled artisan can employ known techniques of protein engineering that use recombinantly expressing cells. (See, e.g., Link et al., Non-canonical amino acids in protein engineering, Current Opinion in Biotechnology, 14(6):603-609 (2003)). The term “non-canonical amino acid residue” refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit), N^(α)-methylcitrulline (NMeCit), N^(α)-methylhomocitrulline (N^(α)-MeHoCit), ornithine (Orn), N^(α)-Methylornithine (N^(α)-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), N^(α)-methylarginine (NMeR), N^(α)-methylleucine (N^(α)-MeL or NMeL), N-methylhomolysine (NMeHoK), N^(α)-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated herein “K(N^(ε)-glycyl)” or “K(glycyl)” or “K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methyl leucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other similar amino acids, and derivatized forms of any of these as described herein. Table 1B contains some exemplary non-canonical amino acid residues that are useful in accordance with the present invention and associated abbreviations as typically used herein, although the skilled practitioner will understand that different abbreviations and nomenclatures may be applicable to the same substance and my appear interchangeably herein.

Table 1B. Useful non-canonical amino acids for amino acid addition, insertion, or substitution into toxin peptide sequences, including OSK1 peptide analog sequences, in accordance with the present invention. In the event an abbreviation listed in Table 1B differs from another abbreviation for the same substance disclosed elsewhere herein, both abbreviations are understood to be applicable.

Abbreviation Amino Acid Sar Sarcosine Nle norleucine Ile isoleucine 1-Nal 3-(1-naphthyl)alanine 2-Nal 3-(2-naphthyl)alanine Bip 4,4′-biphenyl alanine Dip 3,3-diphenylalanine Nvl norvaline NMe-Val Nα-methyl valine NMe-Leu Nα-methyl leucine NMe-Nle Nα-methyl norleucine Cpg cyclopentyl glycine Chg cyclohexyl glycine Hyp hydroxy proline Oic Octahydroindole-2-Carboxylic Acid Igl Indanyl glycine Aib aminoisobutyric acid Aic 2-aminoindane-2-carboxylic acid Pip pipecolic acid BhTic β-homo Tic BhPro β-homo proline Sar Sarcosine Cpg cyclopentyl glycine Tiq 1,2,3,4-L-Tetrahydroisoquinoline-1-Carboxylic acid Nip Nipecotic Acid Thz Thiazolidine-4-carboxylic acid Thi 3-thienyl alanine 4GuaPr 4-guanidino proline 4Pip 4-Amino-1-piperidine-4-carboxylic acid Idc indoline-2-carboxylic acid Hydroxyl-Tic 1,2,3,4-Tetrahydroisoquinoline-7-hydroxy-3- carboxylic acid Bip 4,4′-biphenyl alanine Ome-Tyr O-methyl tyrosine I-Tyr Iodotyrosine Tic 1,2,3,4-L-Tetrahydroisoquinoline-3-Carboxylic acid Igl Indanyl glycine BhTic β-homo Tic BhPhe β-homo phenylalanine AMeF α-methyl Phenyalanine BPhe β-phenylalanine Phg phenylglycine Anc 3-amino-2-naphthoic acid Atc 2-aminotetraline-2-carboxylic acid NMe-Phe Nα-methyl phenylalanine NMe-Lys Nα-methyl lysine Tpi 1,2,3,4-Tetrahydronorharman-3-Carboxylic acid Cpg cyclopentyl glycine Dip 3,3-diphenylalanine 4Pal 4-pyridinylalanine 3Pal 3-pyridinylalanine 2Pal 2-pyridinylalanine 4Pip 4-Amino-1-piperidine-4-carboxylic acid 4AmP 4-amino-phenylalanine Idc indoline-2-carboxylic acid Chg cyclohexyl glycine hPhe homophenylalanine BhTrp β-homotryptophan pI-Phe 4-iodophenylalanine Aic 2-aminoindane-2-carboxylic acid NMe-Lys Nα-methyl lysine Orn ornithine Dpr 2,3-Diaminopropionic acid Dbu 2,4-Diaminobutyric acid homoLys homolysine N-eMe-K Nε-methyl-lysine N-eEt-K Nε-ethyl-lysine N-eIPr-K Nε-isopropyl-lysine bhomoK β-homolysine rLys Lys ψ(CH₂NH)-reduced amide bond rOrn Orn ψ(CH₂NH)-reduced amide bond Acm acetamidomethyl Ahx 6-aminohexanoic acid εAhx 6-aminohexanoic acid K(NPeg11) Nε-(O-(aminoethyl)-O′-(2-propanoyl)- undecaethyleneglycol)-Lysine K(NPeg27) Nε-(O-(aminoethyl)-O′-(2-propanoyl)- (ethyleneglycol)27-Lysine Cit Citrulline hArg homoarginine hCit homocitrulline NMe-Arg Nα-methyl arginine (NMeR) Guf 4-guanidinyl phenylalanine bhArg β-homoarginine 3G-Dpr 2-amino-3-guanidinopropanoic acid 4AmP 4-amino-phenylalanine 4AmPhe 4-amidino-phenylalanine 4AmPig 2-amino-2-(1-carbamimidoylpiperidin-4- yl) acetic acid 4GuaPr 4-guanidino proline N-Arg Nα-[(CH₂)₃NHCH(NH)NH₂] substituted glycine rArg Arg ψ(CH₂NH) -reduced amide bond 4PipA 4-Piperidinyl alanine NMe-Arg Nα-methyl arginine (or NMeR) NMe-Thr Nα-methyl threonine(or NMeThr)

Nomenclature and Symbolism for Amino Acids and Peptides by the UPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) have been published in the following documents: Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69].

As stated herein, in accordance with the present invention, peptide portions of the inventive compositions, such as the toxin peptide or a peptide linker, can also be chemically derivatized at one or more amino acid residues. Peptides that contain derivatized amino acid residues can be synthesized by known organic chemistry techniques. “Chemical derivative” or “chemically derivatized” in the context of a peptide refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty canonical amino acids, whether in L- or D-form. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine maybe substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

Useful derivatizations include, in some embodiments, those in which the amino terminal of the toxin peptide, such as but not limited to the OSK1 peptide analog, is chemically blocked so that conjugation with the vehicle will be prevented from taking place at an N-terminal free amino group. There may also be other beneficial effects of such a modification, for example a reduction in the toxin peptide's susceptibility to enzymatic proteolysis. The N-terminus of the toxin peptide, e.g., the OSK1 peptide analog, can be acylated or modified to a substituted amine, or derivatized with another functional group, such as an aromatic or aryl moiety (e.g., an indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn₂), benzoyl, or benzyloxycarbonyl (Cbz or Z)), N,N-dimethylglycine or creatine. For example, in some embodiments, an acyl moiety, such as, but not limited to, a formyl, acetyl (Ac), propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, or nonanoyl, can be covalently linked to the N-terminal end of the peptide, e.g., the OSK1 peptide analog, which can prevent undesired side reactions during conjugation of the vehicle to the peptide. Alternatively, a fatty acid (e.g. butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic or the like) or polyethylene glycol moiety can be covalently linked to the N-terminal end of the peptide, e.g., the OSK1 peptide analog. Other exemplary N-terminal derivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or benzyloxycarbonyl-NH-(Cbz-NH—), wherein R and R¹ are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.

In some embodiments of the present invention, basic residues (e.g., lysine) of the toxin peptide of interest can be replaced with other residues (nonfunctional residues preferred). Such molecules will be less basic than the molecules from which they are derived and otherwise retain the activity of the molecules from which they are derived, which can result in advantages in stability and immunogenicity; the present invention should not, however, be limited by this theory.

Additionally, physiologically acceptable salts of the inventive compositions are also encompassed, including when the inventive compositions are referred to herein as “molecules” or “compounds.”. By “physiologically acceptable salts” is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some non-limiting examples of pharmaceutically acceptable salts are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; maleate; tartrate; glycolate; gluconate; succinate; mesylate; besylate; salts of gallic acid esters (gallic acid is also known as 3, 4, 5 trihydroxybenzoic acid) such as PentaGalloylGlucose (PGG) and epigallocatechin gallate (EGCG), salts of cholesteryl sulfate, pamoate, tannate and oxalate salts.

Structure of Compounds:

In general. Recombinant proteins have been developed as therapeutic agents through, among other means, covalent attachment to half-life extending moieties. Such moieties include the “Fc” domain of an antibody, as is used in Enbrel® (etanercept), as well as biologically suitable polymers (e.g., polyethylene glycol, or “PEG”), as is used in Neulasta® (pegfilgrastim). Feige et al. described the use of such half-life extenders with peptides in U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 (hereby incorporated by reference in its entirety).

The present inventors have determined that molecules of this invention—peptides of about 80 amino acids or less with at least two intrapeptide disulfide bonds—possess therapeutic advantages when covalently attached to half-life extending moieties. Molecules of the present invention can further comprise an additional pharmacologically active, covalently bound peptide, which can be bound to the half-life extending moiety (F¹ and/or F²) or to the peptide portion (P). Embodiments of the inventive compositions containing more than one half-life extending moiety (F¹ and F²) include those in which F¹ and F² are the same or different half-life extending moieties. Examples (with or without a linker between each domain) include structures as illustrated in FIG. 75 as well as the following embodiments (and others described herein and in the working Examples):

20KPEG—toxin peptide—Fc domain, consistent with the formula [(F¹)₁—(X²)₁—(F²)₁];

20KPEG—toxin peptide—Fc CH2 domain, consistent with the formula [(F¹)₁—(X²)₁—(F²)₁];

20KPEG—toxin peptide—HSA, consistent with the formula [(F¹)₁(X²)₁—(F²)₁];

20KPEG—Fc domain—toxin peptide, consistent with the formula [(F¹)₁—(F²)₁—(X³)₁];

20KPEG—Fc CH2 domain—toxin peptide, consistent with the formula [(F¹)₁—(F²)₁—(X³)₁]; and

20KPEG—HSA—toxin peptide, consistent with the formula [(F¹)₁—(F²)₁—(X³)₁].

Toxin peptides. Any number of toxin peptides (i.e., “P”, or equivalently shown as “P¹” in FIG. 2) can be used in conjunction with the present invention. Of particular interest are the toxin peptides ShK, HmK, MgTx, AgTx2, Agatoxins, and HsTx1, as well as modified analogs of these, in particular OsK1 (also referred to as “OSK1”) peptide analogs of the present invention, and other peptides that mimic the activity of such toxin peptides. As stated herein above, if more than one toxin peptide “P” is present in the inventive composition, “P” can be independently the same or different from any other toxin peptide(s) also present in the inventive composition. For example, in a composition having the formula P-(L)_(g)-F¹-(L)_(f)-P, both of the toxin peptides, “P”, can be the same peptide analog of ShK, different peptide analogs of ShK, or one can be a peptide analog of ShK and the other a peptide analog of OSK1. In a preferred embodiment, at least one P is a an OSK1 peptide analog as further described herein.

In some embodiments of the invention, other peptides of interest are especially useful in molecules having additional features over the molecules of structural Formula I. In such molecules, the molecule of Formula I further comprises an additional pharmacologically active, covalently bound peptide, which is an agonistic peptide, an antagonistic peptide, or a targeting peptide; this peptide can be conjugated to F¹ or F² or P. Such agonistic peptides have activity agonistic to the toxin peptide but are not required to exert such activity by the same mechanism as the toxin peptide. Peptide antagonists are also useful in embodiments of the invention, with a preference for those with activity that can be complementary to the activity of the toxin peptide. Targeting peptides are also of interest, such as peptides that direct the molecule to particular cell types, organs, and the like. These classes of peptides can be discovered by methods described in the references cited in this specification and other references. Phage display, in particular, is useful in generating toxin peptides for use in the present invention. Affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), J. Receptor and Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference in its entirety. Such proteins of interest are preferred for use in this invention.

Particularly preferred peptides appear in the following tables. These peptides can be prepared by methods disclosed in the art or as described hereinafter. Single letter amino acid abbreviations are used. Unless otherwise specified, each X is independently a nonfunctional residue.

TABLE 1 Kv1.3 inhibitor peptide sequences Short-hand SEQ Sequence/structure designation ID NO: LVKCRGTSDCGRPCQQQTGCPNSKCIN Pi1 21 RMCKCYGC TISCTNPKQCYPHCKKETGYPNAKCMNR Pi2 17 KCKCFGR TISCTNEKQCYPHCKKETGYPNAKCMNR Pi3 18 KCKCFGR IEAIRCGGSRDCYRPCQKRTGCPNAKCIN Pi4 19 KTCKCYGCS ASCRTPKDCADPCRKETGCPYGKCMNR HsTx1 61 KCKCNRC GVPINVSCTGSPQCIKPCKDAGMRFGKC AgTx2 23 MNRKCHCTPK GVPINVKCTGSPQCLKPCKDAGMRFGKC AgTx1 85 INGKCHCTPK GVIINVKCKISRQCLEPCKKAGMRFGKCMN OSK1 25 GKCHCTPK ZKECTGPQHCTNFCRKNKCTHGKCMNRK Anuroctoxin 62 CKCFNCK TIINVKCTSPKQCSKPCKELYGSSAGAKC NTX 30 MNGKCKCYNN TVIDVKCTSPKQCLPPCKAQFGIRAGAKC HgTx1 27 MNGKCKCYPH QFTNVSCTTSKECWSVCQRLHNTSRGKC ChTx 36 MNKKCRCYS VFINAKCRGSPECLPKCKEAIGKAAGKCM Titystoxin-Ka 86 NGKCKCYP VCRDWFKETACRHAKSLGNCRTSQKYRA BgK 9 NCAKTCELC VGINVKCKHSGQCLKPCKDAGMRFGKCIN BmKTX 26 GKCDCTPKG QFTDVKCTGSKQCWPVCKQMFGKPNGKC BmTx1 40 MNGKCRCYS VFINVKCRGSKECLPACKAAVGKAAGKCM Tc30 87 NGKCKCYP TGPQTTCQAAMCEAGCKGLGKSMESCQG Tc32 13 DTCKCKA

TABLE 2 ShK peptide and ShK peptide analog sequences Short-hand SEQ Sequence/structure designation ID NO: RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK 5 RSCIDTIPKSRCTAFQSKHSMKYRLSFCRKTSGTC ShK-S17/S32 88 RSSIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTS ShK-S3/S35 89 SSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-S1 90 (N-acetylarg) ShK-N-acetylarg1 91 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-d1 92 CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-d2 93 ASCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A1 94 RSCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A4 95 RSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A4/A15 96 RSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A4/A15/A25 97 RSCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A6 98 RSCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A7 99 RSCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A8 100 RSCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A9 101 RSCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC ShK-E9 102 RSCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q9 103 RSCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC ShK-A10 104 RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC ShK-A11 105 RSCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC ShK-E11 106 RSCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q11 107 RSCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC ShK-A13 108 RSCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A15 109 RSCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC ShK-W15 110 RSCIDTIPKSRCTAX^(s15)QCKHSMKYRLSFCRKTCGTC ShK-X15 111 RSCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A15/A25 112 RSCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC ShK-A16 113 RSCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC ShK-E16 114 RSCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC ShK-A18 115 RSCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC ShK-E18 116 RSCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC ShK-A19 117 RSCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC ShK-K19 118 RSCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC ShK-A20 119 RSCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC ShK-A21 120 RSCIDTIPKSRCTAFQCKHSX^(s21)KYRLSFCRKTCGTC ShK-X21 121 RSCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC ShK-Nle21 122 RSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A22 123 RSCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC ShK-E22 124 RSCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC ShK-R22 125 RSCIDTIPKSRCTAFQCKHSMX^(s22)YRLSFCRKTCGTC ShK-X22 126 RSCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC ShK-Nle22 127 RSCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC ShK-Orn22 128 RSCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC ShK-Homocit22 129 RSCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLS ShK-Diamino- 130 FCRKTCGTC propionic22 RSCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC ShK-A23 131 RSCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC ShK-S23 132 RSCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC ShK-F23 133 RSCIDTIPKSRCTAFQCKHSMKX^(s23)RLSFCRKTCGTC ShK-X23 134 RSCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC ShK-Nitrophe23 135 RSCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC ShK-Aminophe23 136 RSCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCG ShK-Benzylphe23 137 TC RSCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC ShK-A24 138 RSCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC ShK-E24 139 RSCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC ShK-A25 140 RSCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC ShK-A26 141 RSCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC ShK-A27 142 RSCIDTIPKSRCTAFQCKHSMKYRLSX^(s27)CRKTCGTC ShK-X27 143 RSCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC ShK-A29 144 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC ShK-A30 145 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC ShK-A31 146 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC ShK-A34 147 SCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A4d1 148 SCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A4/A15d1 149 SCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A4/A15/A25 150 d1 SCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A6 d1 151 SCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A7 d1 152 SCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A8 d1 153 SCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC ShK-A9 d1 154 SCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC ShK-E9 d1 155 SCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q9 d1 156 SCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC ShK-A10 d1 157 SCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC ShK-A11 d1 158 SCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC ShK-E11 d1 159 SCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q11 d1 160 SCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC ShK-A13 d1 161 SCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK-A15 d1 162 SCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC ShK-W15 d1 163 SCIDTIPKSRCTAX^(s15)QCKHSMKYRLSFCRKTCGTC ShK-X15 d1 164 SCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK-A15/A25 d1 165 SCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC ShK-A16 d1 166 SCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC ShK-E16 d1 167 SCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC ShK-A18 d1 168 SCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC ShK-E18 d1 169 SCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC ShK-A19 d1 170 SCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC ShK-K19 d1 171 SCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC ShK-A20 d1 172 SCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC ShK-A21 d1 173 SCIDTIPKSRCTAFQCKHSX^(s21)KYRLSFCRKTCGTC ShK-X21 d1 174 SCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC ShK-Nle21 d1 175 SCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A22 d1 176 SCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC ShK-E22 d1 177 SCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC ShK-R22 d1 178 SCIDTIPKSRCTAFQCKHSMX^(s22)YRLSFCRKTCGTC ShK-X22 d1 179 SCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC ShK-Nle22 d1 180 SCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC ShK-Orn22 d1 181 SCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC ShK-Homocit22 182 d1 SCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLSF ShK-Diamino- 183 CRKTCGTC propionic22 d1 SCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC ShK-A23 d1 184 SCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC ShK-S23 d1 185 SCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC ShK-F23 d1 186 SCIDTIPKSRCTAFQCKHSMKX^(s23)RLSFCRKTCGTC ShK-X23 d1 187 SCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC ShK-Nitrophe23 188 d1 SCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC ShK-Aminophe23 189 d1 SCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCGTC ShK-Benzylphe23 190 d1 SCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC ShK-A24 d1 191 SCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC ShK-E24 d1 192 SCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC ShK-A25 d1 193 SCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC ShK-A26 d1 194 SCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC ShK-A27 d1 195 SCIDTIPKSRCTAFQCKHSMKYRLSX^(s27)CRKTCGTC ShK-X27 d1 196 SCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC ShK-A29 d1 197 SCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC ShK-A30 d1 198 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC ShK-A31 d1 199 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC ShK-A34 d1 200 YSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Y1 548 KSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-K1 549 HSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-H1 550 QSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-Q1 551 PPRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC PP-ShK 552 MRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC M-ShK 553 GRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC G-ShK 554 YSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-Y1/A22 555 KSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-K1/A22 556 HSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-H1/A22 557 QSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-Q1/A22 558 PPRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC PP-ShK-A22 559 MRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC M-ShK-A22 560 GRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC G-ShK-A22 561 RSCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A22 884 SCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A22 d1 885 RSCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-V9 886 RSCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A22 887 SCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC ShK-V9 d1 888 SCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A22 d1 889 RSCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC ShK-E9/A22 890 SCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC ShK-E9/A22 d1 891 RSCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC ShK-A11/A22 892 SCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC ShK-A11/22 d1 893 RSCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC ShK-E11/A22 894 SCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC ShK-E11/A22 d1 895 RSCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC ShK-D14 896 RSCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC ShK-D14/A22 897 SCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC ShK-D14 d1 898 SCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC ShK-D14/A22 d1 899 RSCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK-A15A/22 900 SCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK-A15/A22 d1 901 RSCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC ShK-I15 902 RSCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC ShK-I15/A22 903 SCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC ShK-I15 d1 904 SCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC ShK-I15/A22 d1 905 RSCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC ShK-V15 906 RSCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC ShK-V15/A22 907 SCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC ShK-V15 d1 908 SCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC ShK-V15/A22 d1 909 RSCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC ShK-R16 910 RSCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC ShK-R16/A22 911 SCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC ShK-R16 d1 912 SCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC ShK-R16/A22 d1 913 RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC ShK-K16 914 RSCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC ShK-K16/A22 915 SCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC ShK-K16 d1 916 SCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC ShK-K16/A22 d1 917 RSCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/E11 918 RSCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/E11/A22 919 SCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/E11 d1 920 SCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/E11/A22 921 d1 RSCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/E11 922 RSCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/E11/A22 923 SCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/E11 d1 924 SCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/E11/A22 925 d1 RSCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/A11 926 RSCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A11/A22 927 SCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC ShK-V9/A11 d1 928 SCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC ShK-V9/A11/A22 929 d1 RSCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/A11 930 RSCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A11/A22 931 SCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC ShK-A9/A11 d1 932 SCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC ShK-A9/A11/A22 933 d1 RSCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 934 E11/D14/I15/R16 RSCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 935 E11/D14/I15/R16/ A22 SCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 936 E11/D14/I15/R16 d1 SCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 937 E11/D14/I15//R16 A22 d1 RSCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 938 V9/E11/D14/I15/ R16 RSCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 939 V9/E11/D14/I15/ R16/A22 SCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 940 V9/E11/D14/I15/ R16 d1 SCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 941 V9/E11/D14/I15/ R16/A22 d1 RSCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC ShK- 942 V9/E11/D14/I15 RSCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC ShK- 943 V9/E11/D14/I15/A22 SCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC ShK- 944 V9/E11/D14/I15 d1 SCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC ShK- 945 V9/E11/D14/I15/A22 d1 RTCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 946 T2/K4/L6/V9/E11/ D14/I15/R16 RTCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 947 T2/K4/L6/V9/E11/ D14/I15/R16/A22 TCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC ShK- 948 T2/K4/L6/V9/E11/ D14/I15/R16 d1 TCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC ShK- 949 T2/K4/L6/V9/E11/ D14/I15/R16/A22 d1 (L-Phosphotyrosine)- ShK(L5) 950 AEEARSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC QSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC ShK Q1/A4/A15 1295 QSCADTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC ShK 1296 Q1/A4/A15/A22 QSCADTIPKSRCTAAQCKHSM(Dap)YRLSFCRKTCGTC ShK 1297 Q1/A4/A15/Dap22 QSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC ShK 1298 Q1/A4/A15/A25 QSCADTIPKSRCTAAQCKHSMAYRASFCRKTCGTC ShK 1299 Q1/A4/A15/A22/A25 QSCADTIPKSRCTAAQCKHSM(Dap)YRASFCRKTCGTC ShK 1300 Q1/A4/A15/Dap22/ A25

Many peptides as described in Table 2 can be prepared as described in U.S. Pat. No. 6,077,680 issued Jun. 20, 2000 to Kem et al., which is hereby incorporated by reference in its entirety. Other peptides of Table 2 can be prepared by techniques known in the art. For example, ShK(L5) (SEQ ID NO: 950) can be prepared as described in Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4): 1369-81 (2005), which is hereby incorporated by reference in its entirety. In Table 2 and throughout the specification, X^(s15), X^(s21), X^(s22), X^(s23) and X^(s27) each independently refer to nonfunctional amino acid residues.

TABLE 3 HmK, BgK, AeK and AsKS peptide and peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK 6 ATCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-A1 201 STCKDLIPVSECTDIRCRTSMKYRLNLCRK HmK-S1 202 TCGSC TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1 203 SCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/S2 204 TCIDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/I4 205 TCKDTIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/T6 206 TCKDLIPKSECTDIRCRTSMKYRLNLCRKTCGSC HmK-d1/K9 207 TCKDLIPVSRCTDIRCRTSMKYRLNLCRKTCGSC HmK- 208 d1/R11 TCKDLIPVSECTAIRCRTSMKYRLNLCRKTCGSC HmK- 209 d1/A14 TCKDLIPVSECTDFRCRTSMKYRLNLCRKTCGSC HmK- 210 d1/F15 TCKDLIPVSECTDIQCRTSMKYRLNLCRKTCGSC HmK- 211 d1/Q16 TCKDLIPVSECTDIRCKTSMKYRLNLCRKTCGSC HmK- 212 d1/K18 TCKDLIPVSECTDIRCRHSMKYRLNLCRKTCGSC HmK- 213 d1/H19 TCKDLIPVSECTDIRCRTSMKYRLSLCRKTCGSC HmK- 214 d1/S26 TCKDLIPVSECTDIRCRTSMKYRLNFCRKTCGSC HmK- 215 d1/F27 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGTC HmK- 216 d1/T34 TCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC HmK- 217 d1/R11/F27 ATCKDLIPVSRCTDIRCRTSMKYRLNFCRKT HmK- 218 CGSC A1/R11/F27 TCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGSC HmK-d1/Z1 219 TCIDTIPKSRCTAFQCRTSMKYRLNFCRKTCGSC HmK-d1/Z2 220 TCADLIPASRCTAIACRTSMKYRLNFCRKTCGSC HmK-d1/Z3 221 TCADLIPASRCTAIACKHSMKYRLNFCRKTCGSC HmK-d1/Z4 222 TCADLIPASRCTAIACAHSMKYRLNFCRKTCGSC HmK-d1/Z5 223 RTCKDLIPVSECTDIRCRTSMX^(h22) HmK-X22 224 YRLNLCRKTCGSC ATCKDLX^(h6)PVSRCTDIRCRTSMKX^(h22)RLNX^(h26) HmK-X6, 225 CRKTCGSC 22, 26 VCRDWFKETACRHAKSLGNCRTSQKYRANCA BgK 9 KTCELC ACRDWFKETACRHAKSLGNCRTSQKYRANCA BgK-A1 226 KTCELC VCADWFKETACRHAKSLGNCRTSQKYRANCA BgK-A3 227 KTCELC VCRDAFKETACRHAKSLGNCRTSQKYRANCA BgK-A5 228 KTCELC VCRDWFKATACRHAKSLGNCRTSQKYRANCA BgK-A8 229 KTCELC VCRDWFKEAACRHAKSLGNCRTSQKYRANCA BgK-A9 230 KTCELC VCRDWFKETACAHAKSLGNCRTSQKYRANCA BgK-A12 231 KTCELC VCRDWFKETACRHAASLGNCRTSQKYRANCA BgK-A15 232 KTCELC VCRDWFKETACRHAKALGNCRTSQKYRANCA BgK-A16 233 KTCELC VCRDWFKETACRHAKSAGNCRTSQKYRANCA BgK-A17 234 KTCELC VCRDWFKETACRHAKSLGNCATSQKYRANCA BgK-A21 235 KTCELC VCRDWFKETACRHAKSLGNCRASQKYRANCA BgK-A22 236 KTCELC VCRDWFKETACRHAKSLGNCRTSQKYAANCA BgK-A27 237 KTCELC VCRDWFKETACRHAKSLGNCRTSQKYRANCA BgK-A32 238 ATCELC VCRDWFKETACRHAKSLGNCRTSQKYRANCA BgK-A33 239 KACELC VCRDWFKETACRHAKSLGNCRTSQKYRANCA BgK-A35 240 KTCALC VCRDWFKETACRHAKSLGNCRTSQKYRANCA BgK-A37 241 KTCEAC GCKDNFSANTCKHVKANNNCGSQKYATNCA AeK 7 KTCGKC ACKDNFAAATCKHVKENKNCGSQKYATNCA AsKS 8 KTCGKC

In Table 3 and throughout the specification, X^(h6), X^(h22), X^(h26) are each independently nonfunctional residues.

TABLE 4 MgTx peptide and MgTx peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx 28 CKCYPH TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-A6 242 CKCYPH TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-S6 243 CKCYPH TIINVKCTSPAQCLPPCKAQFGQSAGAKCMNGK MgTx-A11 244 CKCYPH TIINVKCTSPKQCLPPCAAQFGQSAGAKCMNGK MgTx-A18 245 CKCYPH TIINVKCTSPKQCLPPCKAQFGQSAGAACMNGK MgTx-A28 246 CKCYPH TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGA MgTx-A33 247 CKCYPH TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-A35 248 CACYPH TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-H39N 249 CKCYPN TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx- 250 CKCYPN A6/H39N TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx- 251 CKCYS S6/38/d39 TIITISCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-T4/I5/S6 252 CKCYPH TISCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx- 253 CKCYPH d3/T4/I5/S6 TISCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-Pi2 254 CKCFGR NVACTSPKQCLPPCKAQFGQSAGAKCMNG MgTx-d3/A6 255 KCKCYPH QFTNVSCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-ChTx 256 CKCYS QFTDVDCTSPKQCLPPCKAQFGQSAGAKCM MgTx-IbTx 257 NGKCKCYQ IINVSCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-Z1 258 CKCYPH IITISCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-Z2 259 CKCYPH GVIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGK MgTx-Z3 260 CKCYPH

Many peptides as described in Table 4 can be prepared as described in WO 95/03065, published Feb. 2, 1995, for which the applicant is Merck & Co., Inc. That application corresponds to U.S. Ser. No. 07/096,942, filed 22 Jul. 1993, which is hereby incorporated by reference in its entirety.

TABLE 5 AgTx2 peptide and AgTx2 peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: GVPINVSCTGSPQCIKPCKDAGMR AgTx2 23 FGKCMNRKCHCTPK GVPIAVSCTGSPQCIKPCKDAGMR AgTx2-A5 261 FGKCMNRKCHCTPK GVPINVSCTGSPQCIAPCKDAGMR AgTx2-A16 262 FGKCMNRKCHCTPK GVPINVSCTGSPQCIKPCADAGMR AgTx2-A19 263 FGKCMNRKCHCTPK GVPINVSCTGSPQCIKPCKDAGMA AgTx2-A24 264 FGKCMNRKCHCTPK GVPINVSCTGSPQCIKPCKDAGMR AgTx2-A27 265 FGACMNRKCHCTPK GVPINVSCTGSPQCIKPCKDAGMR AgTx2-A31 266 FGKCMNAKCHCTPK GVPINVSCTGSPQCIKPCKDAGMR AgTx2-A32 267 FGKCMNRACHCTPK GVPINVSCTGSPQCIKPCKDAGMR AgTx2-A38 268 FGKCMNRKCHCTPA GVPIAVSCTGSPQCIKPCKDAGMR AgTx2-A5/A38 269 FGKCMNRKCHCTPA GVPINVSCTGSPQCIKPCKDAGMR AgTx2-G31 270 FGKCMNGKCHCTPK GVPIIVSCKGSRQCIKPCKDAGMR AgTx2- 271 FGKCMNGKCHCTPK OSK_z1 GVPIIVSCKISRQCIKPCKDAGMR AgTx2- 272 FGKCMNGKCHCTPK OSK_z2 GVPIIVKCKGSRQCIKPCKDAGMR AgTx2- 273 FGKCMNGKCHCTPK OSK_z3 GVPIIVKCKISRQCIKPCKDAGMR AgTx2- 274 FGKCMNGKCHCTPK OSK_z4 GVPIIVKCKISRQCIKPCKDAGMR AgTx2- 275 FGKCMNGKCHCTPK OSK_z5

TABLE 6 Heteromitrus spinnifer (HsTx1) peptide and HsTx1 peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1 61 ASCXTPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-X4 276 ASCATPKDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-A4 277 ASCRTPXDCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-X7 278 ASCRTPADCADPCRKETGCPYGKCMNRKCKCNRC HsTx1-A7 279 ASCRTPKDCADPCXKETGCPYGKCMNRKCKCNRC HsTx1-X14 280 ASCRTPKDCADPCAKETGCPYGKCMNRKCKCNRC HsTx1-A14 281 ASCRTPKDCADPCRXETGCPYGKCMNRKCKCNRC HsTx1-X15 282 ASCRTPKDCADPCRAETGCPYGKCMNRKCKCNRC HsTx1-A15 283 ASCRTPKDCADPCRKETGCPYGXCMNRKCKCNRC HsTx1-X23 284 ASCRTPKDCADPCRKETGCPYGACMNRKCKCNRC HsTx1-A23 285 ASCRTPKDCADPCRKETGCPYGKCMNXKCKCNRC HsTx1-X27 286 ASCRTPKDCADPCRKETGCPYGKCMNAKCKCNRC HsTx1-A27 287 ASCRTPKDCADPCRKETGCPYGKCMNRXCKCNRC HsTx1-X28 288 ASCRTPKDCADPCRKETGCPYGKCMNRACKCNRC HsTx1-A28 289 ASCRTPKDCADPCRKETGCPYGKCMNRKCXCNRC HsTx1-X30 290 ASCRTPKDCADPCRKETGCPYGKCMNRKCACNRC HsTx1-A30 291 ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNXC HsTx1-X33 292 ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNAC HsTx1-A33 293

Peptides as described in Table 5 can be prepared as described in U.S. Pat. No. 6,689,749, issued Feb. 10, 2004 to Lebrun et al., which is hereby incorporated by reference in its entirety.

TABLE 7 Orthochirus scrobiculosus (OSK1) peptide and OSK1 peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1 25 GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1-S7 1303 GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK OSK1-K16 294 GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK OSK1-D20 295 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-K16, D20 296 GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-S7, K16, D20 1308 GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-P12, K16, D20 297 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK OSK1-K16, D20, Y36 298 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-P12, 562 K16, D20 GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-P12, K16, 563 D20-NH₂ Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-P12, 564 K16, D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH₂ OSK1-K16, D20, 565 Y36-NH₂ Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK Ac-OSK1-K16, 566 D20, Y36 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH₂ Ac-OSK1-K16, D20, 567 Y36-NH₂ GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K16-NH₂ 568 Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1-K16 569 Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16-NH₂ 570 Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-D20 571 GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-D20-NH₂ 572 Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-D20-NH₂ 573 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-NH₂ 574 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1 575 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-NH₂ 576 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K16, D20-NH₂ 577 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-K16, 578 D20 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16, D20- 579 NH₂ VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Δ1-OSK1 580 Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1 581 VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-NH₂ 582 Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-NH₂ 583 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK OSK1-A34 584 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Ac-OSK1-A34 585 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ OSK1-A34-NH₂ 586 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-A34-NH₂ 587 VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Δ1-OSK1-K16, D20 588 Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1-K16, 589 D20 VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-K16, D20- 590 NH₂ Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-K16, 591 D20-NH₂ NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-4)-OSK1-K16, 592 D20 Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-4)-OSK1- 593 K16, D20 NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-4)-OSK1-K16, 594 D20-NH₂ Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-4)-OSK1- 595 K16, D20-NH₂ KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-6)-OSK1-K16, 596 D20 Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-6)-OSK1- 597 K16, D20 KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-6)-OSK1-K16, 598 D20-NH₂ Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-6)-OSK1- 599 K16, D20-NH₂ CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (Δ1-7)-OSK1-K16, 600 D20 Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-(Δ1-7)-OSK1- 601 K16, D20 CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ (Δ1-7)-OSK1-K16, 602 D20-NH₂ Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-(Δ1-7)-OSK1- 603 K16, D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK OSK1-K16, D20, 604 N25 GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH₂ OSK1-K16, D20, 605 N25-NH₂ Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK Ac-OSK1-K16, 606 D20, N25 Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH₂ Ac-OSK1-K16, D20, 607 N25-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK OSK1-K16, D20, 608 R31 GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH₂ OSK1-K16, D20, 609 R31-NH₂ Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK Ac-OSK1-K16, 610 D20, R31 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH₂ Ac-OSK1-K16, D20, 611 R31-NH₂ GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK OSK1-K12, K16, 612 R19, D20 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK Ac-OSK1-K12, K16, 613 R19, D20 GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K12, K16, 614 R19, D20-NH₂ Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K12, K16, 615 R19, D20-NH₂ TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Δ1-OSK1-T2, K16, 616 D20 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-Δ1-OSK1-T2, 617 K16, D20 TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Δ1-OSK1-T2, K16, 618 D20-NH₂ Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-Δ1-OSK1-T2, 619 K16, D20-NH₂ GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK OSK1-K3 620 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK Ac-OSK1-K3 621 GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K3-NH₂ 622 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K3-NH₂ 623 GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK OSK1-K3, A34 624 GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK OSK1-K3, K16, D20 625 GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK OSK1-K3, K16, D20, 626 A34 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Ac-OSK1-K3, A34 627 GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ OSK1-K3, A34-NH₂ 628 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-K3, A34- 629 NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK Ac-OSK1-K3, K16, 630 D20, A34 GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH₂ OSK1-K3, K16, D20, 631 A34-NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH₂ Ac-OSK1-K3, K16, 632 D20, A34-NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK Ac-OSK1-K3, K16, 633 D20 GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ OSK1-K3, K16, D20- 634 NH₂ Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH₂ Ac-OSK1-K3, K16, 635 D20-NH₂ GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCT Δ36-38-OSK1-K16, 636 D20 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK OSK1-O16, D20 980 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK OSK1-hLys 981 16, D20 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK OSK1-hArg 982 16, D20 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK OSK1-Cit 16, D20 983 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK OSK1-hCit 984 16, D20 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK OSK1-Dpr 16, D20 985 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK OSK1-Dab 16, D20 986 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK OSK1-O16, D20, Y36 987 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK OSK1-hLys 988 16, D20, Y36 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK OSK1-hArg 989 16, D20, Y36 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK OSK1-Cit 990 16, D20, Y36 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK OSK1-hCit 991 16, D20, Y36 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK OSK1-Dpr 992 16, D20, Y36 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK OSK1-Dab 993 16, D20, Y36 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYPK OSK1- 994 K16, D20, A34, Y36 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYPK OSK1- 995 K16, D20, G34, Y36 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFPK OSK1- 996 K16, D20, A34, F36 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACWPK OSK1- 997 K16, D20, A34, W36 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYPK OSK1- 998 K16, E20, A34, Y36 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK OSK1-O16, D20, A34 999 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK OSK1-hLys 1000 16, D20, A34 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK OSK1-hArg 1001 16, D20, A34 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK OSK1-Cit 1002 16, D20, A34 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK OSK1-hCit 1003 16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK OSK1-Dpr 1004 16, D20, A34 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK OSK1-Dab 1005 16, D20, A34 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1006 O16, D20, GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1007 hLys 16, D20 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1008 hArg 16, D20 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1-Cit 1009 16, D20 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1010 hCit 16, D20 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1-Dpr 1011 16, D20 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1012 Dab16, D20 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1013 O16, D20, A34 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1014 hLys 16, D20, A34 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1- 1015 hArg 16, D20, A34 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1-Cit 1016 16, D20, A34 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC Δ36-38, OSK1- 1017 hCit 16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1-Dpr 1018 16, D20, A34 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC Δ36-38, OSK1-Dab 1019 16, D20, A34 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYGG OSK1- 1020 K16, D20, G34, Y36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG OSK1- 1021 O16, D20, Y36, G37, G38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG OSK1-hLys 1022 16, D20, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG OSK1-hArg 1023 16, D20, Y36, G37, G38 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG OSK1-Cit 1024 16, D20, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG OSK1-hCit 1025 16, D20, Y36, G37, G38 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG OSK1-Dpr 1026 16, D20, Y36, G37, G38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG OSK1- 1027 K16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG OSK1- 1028 O16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG OSK1-hLys 1029 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG OSK1-hArg 1030 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG OSK1-Cit 1031 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG OSK1-hCit 1032 16, D20, A34, Y3, G37, G38 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG OSK1-Dpr 1033 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG OSK1-Dab 1034 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG Δ38, OSK1- 1035 K16, D20, A34, Y36, G37 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG OSK1- 1036 O16, D20, G36-38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG OSK1-hLys 1037 16, D20, G36-38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG OSK1-hArg 1038 16, D20, G36-38 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG OSK1-Cit 1039 16, D20, G36-38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG OSK1-hCit 1040 16, D20, G36-38 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG OSK1-Dpr 1041 16, D20, G36-38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFGG OSK1- 1042 K16, D20, A34, F36, G37, G38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG OSK1- 1043 O16, D20, A34, G36- 38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG OSK1-hLys 1044 16, D20, A34, G36-38 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG OSK1-hArg 1045 16, D20, A34, G36-38 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG OSK1-Cit 1046 16, D20, A34, G36-38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG OSK1-hCit 1047 16, D20, A34, G36-38 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG OSK1-Dpr 1048 16, D20, A34, G36-38 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG OSK1-Dab 1049 16, D20, A34, G36-38 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGG Δ38, OSK1- 1050 K16, D20, A34, G36- 37 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG Δ38, OSK1- 1051 K16, D20, A35, Y36, G37 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGG Δ38, OSK1- 1052 O16, D20, A35, Y36, G37 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK OSK1-hLys 1053 16, E20 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK OSK1-hArg 1054 16, E20 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK OSK1-Cit 16, E20 1055 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK OSK1-hCit 1056 16, E20 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK OSK1-Dpr 16, E20 1057 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK OSK1-Dab 16, E20 1058 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK OSK1-O16, E20, Y36 1059 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK OSK1-hLys 1060 16, E20, Y36 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK OSK1-hArg 1061 16, E20, Y36 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK OSK1-Cit 1062 16, E20, Y36 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK OSK1-hCit 1063 16, E20, Y36 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK OSK1-Dpr 1064 16, E20, Y36 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK OSK1-Dab 1065 16, E20, Y36 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK OSK1-O16, E20, A34 1066 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK OSK1-hLys 1067 16, E20, A34 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK OSK1-hArg 1068 16, E20, A34 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK OSK1-Cit 1069 16, E20, A34 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK OSK1-hCit 1070 16, E20, A34 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK OSK1-Dpr 1071 16, E20, A34 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK OSK1-Dab 1072 16, E20, A34 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1073 O16, E20, GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1074 hLys 16, E20 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1075 hArg 16, E20 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1-Cit 1076 16, E20 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1077 hCit16, E20 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1-Dpr 1078 16, E20 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC Δ36-38, OSK1- 1079 O16, E20, A34 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1- 1080 hLys 16, E20, A34 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1- 1081 hArg 16, E20, A34 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1-Cit 1082 16, E20, A34 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC Δ36-38, OSK1- 1083 hCit 16, E20, A34 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1-Dpr 1084 16, E20, A34 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC Δ36-38, OSK1-Dab 1085 16, E20, A34 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGG OSK1- 1086 K16, E20, Y36, G37, G38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG OSK1- 1087 O16, E20, Y36, G37, G38 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYG Δ38 OSK1- 1088 K16, E20, Y36, G37 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG Δ38 OSK1- 1089 K16, E20, A34, Y36, G37 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG OSK1-hLys 1090 16, E20, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG OSK1-hArg 1091 16, E20, Y36, G37, G38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG OSK1-Cit 1092 16, E20, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG Δ37-38, OSK1- 1093 hCit 16, E20, Y36, G37, G38 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG OSK1-Dpr 1094 16, E20, Y36, G37, G38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG OSK1-Dab 1095 16, E20, Y36, G37, G38 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG Δ38, OSK1- 1096 K16, E20, A34, Y36, G37 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG OSK1- 1097 O16, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG OSK1-hLys 1098 16, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG OSK1-hArg 1099 16, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG OSK1-Cit 1100 16, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG OSK1-hCit 1101 16, E20, A34, Y3, G37, G38 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG OSK1-Dpr 1102 16, E20, A34, Y36, G37, G38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG OSK1-Dab 1103 16, E20, A34, Y36, G37, G38 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACFGG OSK1- 1104 K16, D20, A34, F36, G37, G38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG OSK1- 1105 O16, E20, G36-38 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG OSK1-hLys 1106 16, E20, G36-38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG OSK1-hArg 1107 16, E20, G36-38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG OSK1-Cit 1108 16, E20, G36-38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG OSK1-hCit 1109 16, E20, G36-38 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG OSK1-Dpr 1110 16, E20, G36-38 GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG OSK1- 1111 O16, E20, A34, G36- 38 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG OSK1-hLys 1112 16, E20, A34, G36-38 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG OSK1-hArg 1113 16, E20, A34, G36-38 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG OSK1-Cit 1114 16, E20, A34, G36-38 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP OSK1-hCit 1115 16, E20, A34, G36-38 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTP OSK1-Dpr 1116 16, E20, A34, G36-38 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTP OSK1-Dab 1117 16, E20, A34, G36-38 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK-NH2 OSK1-O16, D20- 1118 amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK- OSK1-hLys 1119 NH2 16, D20-amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK- OSK1-hArg 1120 NH2 16, D20-amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-NH2 OSK1-Cit 1121 16, D20-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK- OSK1-hCit 1122 NH2 16, D20-amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dpr 1123 16, D20-amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dab 16, D20 1124 GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK-NH2 OSK1- 1125 O16, D20, Y36- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK- OSK1-hLys 1126 NH2 16, D20, Y36- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK- OSK1-hArg 1127 NH2 16, D20, Y36- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-NH2 OSK1-Cit 1128 16, D20, Y36- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK- OSK1-hCit 1129 NH2 16, D20, Y36- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-NH2 OSK1-Dpr 1130 16, D20, Y36- amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-NH2 OSK1-Dab 1131 16, D20, Y36- amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK-NH2 OSK1- 1132 O16, D20, A34- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK- OSK1-hLys 1133 NH2 16, D20, A34- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK- OSK1-hArg 1134 NH2 16, D20, A34- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-NH2 OSK1-Cit 1135 16, D20, A34- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACTPK- OSK1-hCit 1136 NH2 16, D20, A34- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-NH2 OSK1-Dpr 1137 16, D20, A34- amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-NH2 OSK1-Dab 1138 16, D20, A34- amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1139 O16, D20, - amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1140 hLys 16, D20- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1141 hArg 16, D20- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-Cit 1142 16, D20-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1143 hCit16, D20- amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-Dpr 1144 16, D20-amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1145 O16, D20, A34- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1146 hLys 16, D20, A34- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1147 hArg 16, D20, A34- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Cit 1148 16, D20, A34- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1149 hCit16, D20, A34 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Dpr 1150 16, D20, A34- amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Dab 1151 16, D20, A34- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1- 1152 O16, D20, Y36, G37, G38- amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1- 1153 O16, D20, Y36, G37, G38 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG- OSK1-hLys 1154 NH2 16, D20, Y36, G37, G38- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG- OSK1-hArg 1155 NH2 16, D20, Y36, G37, G38- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1-Cit 1156 16, D20, Y36, G37, G38- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG- OSK1- 1157 NH2 hCit16, D20, Y36, G37, G38-amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1-Dpr 1158 16, D20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCFGG-NH2 OSK1- 1159 K16, D20, F36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYG-NH2 Δ38-OSK1- 1160 K16, D20, Y36, G37- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG-NH2 Δ38-OSK1- 1161 K16, D20, A34, Y36, G37-amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG-NH2 OSK1- 1162 O16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG- OSK1-hLys 1163 NH2 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG- OSK1-hArg 1164 NH2 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-Cit 1165 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACYGG- OSK1- 1166 NH2 hCit16, D20, A34, Y3, G37, G38-amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-Dpr 1167 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-Dab 1168 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2 OSK1- 1169 K16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG-NH2 OSK1- 1170 O16, D20, G36-38- amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG- OSK1-hLys 1171 NH2 16, D20, G36-38- amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG- OSK1-hArg 1172 NH2 16, D20, G36-38- amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-NH2 OSK1-Cit 1173 16, D20, G36-38- amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG- OSK1- 1174 NH2 hCit16, D20, G36- 38-amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-NH2 OSK1-Dpr 1175 16, D20, G36-38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGG-NH2 OSK1- 1176 K16, D20, A34, G36- 38-amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACFGG-NH2 OSK1- 1177 O16, D20, A34, F36, G37-38-amide GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG-NH2 OSK1- 1178 O16, D20, A34, G36- 38-amide GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG- OSK1-hLys 1179 NH2 16, D20, A34, G36- 38-amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG- OSK1-hArg 1180 NH2 16, D20, A34, G36- 38-amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-Cit 1181 16, D20, A34, G36- 38-amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG- OSK1- 1182 NH2 hCit16, D20, A34, G36- 38-amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-Dpr 1183 16, D20, A34, G36- 38-amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-NH2 OSK1-Dab 1184 16, D20, A34, G36- 38-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-O16, E20- 1185 amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK- OSK1-hLys 1186 NH2 16, E20-amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK- OSK1-hArg 1187 NH2 16, E20-amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-Cit 1188 16, E20-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK- OSK1- 1189 NH2 hCit16, E20- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dpr 1190 16, E20-amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-NH2 OSK1-Dab 1191 16, E20-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK-NH2 OSK1- 1192 O16, E20, Y36- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK- OSK1-hLys 1193 NH2 16, E20, Y36- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK- OSK1-hArg 1194 NH2 16, E20, Y36- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-NH2 OSK1-Cit 1195 16, E20, Y36- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK- OSK1- 1196 NH2 hCit16, E20, Y36- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-NH2 OSK1-Dpr 1197 16, E20, Y36- amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-NH2 OSK1-Dab 1198 16, E20, Y36- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK-NH2 OSK1- 1199 O16, E20, A34- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK- OSK1-hLys 1200 NH2 16, E20, A34- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK- OSK1-hArg 1201 NH2 16, E20, A34- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-NH2 OSK1-Cit 1202 16, E20, A34- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPK- OSK1- 1203 NH2 hCit16, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-NH2 OSK1-Dpr 1204 16, E20, A34- amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-NH2 OSK1-Dab 1205 16, E20, A34- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1206 O16, E20, - amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1207 hLys 16, E20- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1208 hArg 16, E20- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-Cit 1209 16, E20-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1210 hCit16, E20- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1-Dpr 1211 16, E20-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1212 O16, E20, A34- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1213 hLys16, E20, A34- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1- 1214 hArg16, E20, A34- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Cit 1215 16, E20, A34- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2 Δ36-38, OSK1- 1216 hCit16, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Dpr 1217 16, E20, A34- amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC-NH2 Δ36-38, OSK1-Dab 1218 16, E20, A34- amide GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCWGG-NH2 OSK1- 1219 O16, E20, W36, G37, G38-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1- 1220 O16, E20, Y36, G37, G38- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hLys 1221 NH2 16, E20, Y36, G37, G38- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hArg 1222 NH2 16, E20, Y36, G37, G38- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1-Cit 1223 16, E20, Y36, G37, G38- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hCit 1224 NH2 16, E20, Y36, G37, G38- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1-Dpr 1225 16, E20, Y36, G37, G38- amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-NH2 OSK1-Dpr 1226 16, E20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGG-NH2 OSK1- 1227 K16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG-NH2 OSK1- 1228 O16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG- OSK1-hLys 1229 NH2 16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG- OSK1-hArg 1230 NH2 16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-NH2 OSK1-Cit 1231 16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- OSK1-hCit 1232 NH2 16, E20, A34, Y3, G37, G38-amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-NH2 OSK1-Dpr 1233 16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-NH2 OSK1-Dab 1234 16, E20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG-NH2 OSK1- 1235 O16, E20, G36-38- amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG- OSK1-hLys 1236 NH2 16, E20, G36-38- amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG- OSK1-hArg 1237 NH2 16, E20, G36-38- amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-NH2 OSK1-Cit 1238 16, E20, G36-38- amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG- OSK1-hCit 1239 NH2 16, E20, G36-38- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-NH2 OSK1-Dpr 1240 16, E20, G36-38- amide GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG-NH2 OSK1- 1241 O16, E20, A34, G36- 38-amide GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG- OSK1-hLys 1242 NH2 16, E20, A34, G36- 38-amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG- OSK1-hArg 1243 NH2 16, E20, A34, G36- 38-amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG-NH2 OSK1-Cit 1244 16, E20, A34, G36- 38-amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP-NH2 Δ38 OSK1-hCit 1245 16, E20, A34- amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-NH2 OSK1-Dpr 1246 16, E20, A34, G36- 38-amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-NH2 OSK1-Dab 1247 16, E20, A34, G36- 38-amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-NH2 OSK1-K 1248 16, CPA20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-NH2 OSK1-K 1249 16, CPA20, A34, G36- 38-amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACY-NH2 Δ37-38OSK1-K 1250 16, CPA20, A34, Y36- amide Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2 Acetyl-OSK1-K 1251 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG-NH2 OSK1-K 16, 1252 Aad20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-NH2 OSK1-K 16, 1253 Aad20, Y36, G37, G38- amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG OSK1-K 16, 1254 Aad20, A34, Y36, G37, G38 GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG-NH2 OSK1-H 1255 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG OSK1-H 1256 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACY-NH2 Δ37-38-OSK1-H 1257 16, D20, A34, Y36- amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG-NH2 OSK1-H 1258 16, D20, A34, Y36, G37, G38-amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG OSK1-H 1259 16, D20, A34, Y36, G37, G38 GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYPK OSK1-H 1260 16, D20, A34, Y36, GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC Δ36-38 OSK1-H 1261 16, D20, A34, Y36, GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG- OSK1-K 1262 NH2 16, D20, A34, 1Nal36, G37, G38-amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK- OSK1-K 1263 NH2 16, D20, A34, 1Nal36- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG- OSK1-K 1264 NH2 16, D20, A34, 2Nal36, G37, G38-amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-NH2 OSK1-K 1265 16, D20, A34, Cha36, G37, G38-amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[MePhe]GG- OSK1-K 1266 NH2 16, D20, A34, MePhe36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]GG- OSK1-K 1267 NH2 16, D20, A34, BiPhA36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-NH2 OSK1-K 16, D20, 1268 Aib34, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-NH2 OSK1-K 16, D20, 1269 Abu34, Y36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal] Δ37-38 OSK1-H 1270 16, D20, A34, 1Nal36, - amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG- OSK1-H 1271 NH2 16, D20, A34, 1Nal36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-NH2 Δ37-38 OSK1-H 1272 16, D20, A34, 4Bip 36, -amide GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG- OSK1-H 1273 NH2 16, D20, A34, 4Bip 36, G37, G38- amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG OSK1-K16, E20, G36- 1274 38

TABLE 7A Additional useful OSK1 peptide analog sequences SEQ ID NO Sequence 1391 GVIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK 1392 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1393 GVIINVKCKISPQCLKPCKDAGIRFGKCINGKCACTPK 1394 GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACTPK 1395 GGGGSGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1396 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHC 1397 GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCHCTY[Nle] 1398 GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTY[Nle] 1399 GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG 1400 AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1401 GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1402 GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1403 GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1404 GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1405 GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1406 GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1407 GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1408 GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK 1409 GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK 1410 GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1411 GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK 1412 GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK 1413 GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK 1414 GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK 1415 GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK 1416 GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK 1417 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1418 GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK 1419 GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK 1420 GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK 1421 GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK 1422 GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK 1423 GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 1424 GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 1425 GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 1426 GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK 1427 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK 1428 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK 1429 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK 1430 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK 1431 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA 1432 RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1433 GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1434 GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1435 GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1436 GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1437 GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1438 GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1439 GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1440 GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK 1441 GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK 1442 GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK 1443 GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK 1444 GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK 1445 GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK 1446 GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK 1447 GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK 1448 GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK 1449 GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK 1450 GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK 1451 GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK 1452 GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK 1453 GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK 1454 GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK 1455 GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK 1456 GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK 1457 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK 1458 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK 1459 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK 1460 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK 1461 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR 1462 EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1463 GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1464 GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1465 GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1466 GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1467 GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1468 GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1469 GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1470 GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK 1471 GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK 1472 GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK 1473 GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK 1474 GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK 1475 GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK 1476 GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK 1477 GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK 1478 GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK 1479 GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK 1480 GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK 1481 GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK 1482 GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK 1483 GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK 1484 GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK 1485 GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK 1486 GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK 1487 GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK 1488 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK 1489 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK 1490 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK 1491 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK 1492 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE 1493 [1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1494 G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1495 GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1496 GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1497 GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1498 GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1499 GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1500 GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1501 GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHCTPK 1502 GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHCTPK 1503 GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHCTPK 1504 GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHCTPK 1505 GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHCTPK 1506 GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHCTPK 1507 GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHCTPK 1508 GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHCTPK 1509 GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHCTPK 1510 GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHCTPK 1511 GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHCTPK 1512 GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHCTPK 1513 GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHCTPK 1514 GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHCTPK 1515 GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHCTPK 1516 GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHCTPK 1517 GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHCTPK 1518 GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHCTPK 1519 GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHCTPK 1520 GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHCTPK 1521 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]CTPK 1522 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]PK 1523 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-Nal]K 1524 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-Nal] 1525 Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1526 Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1527 Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1528 Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1529 Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1530 Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1531 Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1532 Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1533 Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK 1534 Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK 1535 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1536 Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK 1537 Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK 1538 Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK 1539 Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK 1540 Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK 1541 Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK 1542 Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK 1543 Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK 1544 Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK 1545 Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK 1546 Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK 1547 Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 1548 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 1549 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 1550 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK 1551 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK 1552 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK 1553 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK 1554 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK 1555 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA 1556 Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1557 Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1558 Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1559 Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1560 Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1561 Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1562 Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1563 Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1564 Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK 1565 Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK 1566 Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK 1567 Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK 1568 Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK 1569 Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK 1570 Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK 1571 Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK 1572 Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK 1573 Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK 1574 Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK 1575 Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK 1576 Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK 1577 Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK 1578 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK 1579 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK 1580 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK 1581 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK 1582 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK 1583 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK 1584 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK 1585 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR 1586 Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1587 Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1588 Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1589 Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1590 Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1591 Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1592 Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1593 Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK 1594 Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK 1595 Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK 1596 Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK 1597 Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK 1598 Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK 1599 Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK 1600 Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK 1601 Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK 1602 Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK 1603 Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK 1604 Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK 1605 Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK 1606 Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK 1607 Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK 1608 Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK 1609 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK 1610 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK 1611 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK 1612 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK 1613 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK 1614 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK 1615 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK 1616 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE 1617 Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK 1618 Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK 1619 Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK 1620 Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK 1621 Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK 1622 Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK 1623 Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCH CTPK 1624 Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCH CTPK 1625 Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCH CTPK 1626 Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCH CTPK 1627 Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCH CTPK 1628 Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCH CTPK 1629 Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCH CTPK 1630 Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCH CTPK 1631 Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCH CTPK 1632 Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCH CTPK 1633 Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCH CTPK 1634 Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCH CTPK 1635 Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCH CTPK 1636 Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCH CTPK 1637 Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCH CTPK 1638 Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCH CTPK 1639 Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCH CTPK 1640 Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCH CTPK 1641 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCH CTPK 1642 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCH CTPK 1643 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCH CTPK 1644 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CH CTPK 1645 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC [1-Nal]CTPK 1646 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [1-Nal]PK 1647 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT [1-Nal]K 1648 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP [1-Nal] 1649 AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1650 GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1651 GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1652 GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1653 GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1654 GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1655 GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1656 GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1657 GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1658 GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1659 GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1660 GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK-amide 1661 GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK-amide 1662 GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK-amide 1663 GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK-amide 1664 GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK-amide 1665 GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK-amide 1666 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1667 GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK-amide 1668 GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK-amide 1669 GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK-amide 1670 GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK-amide 1671 GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK-amide 1672 GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 1673 GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 1674 GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 1675 GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK-amide 1676 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK-amide 1677 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-amide 1678 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK-amide 1679 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK-amide 1680 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA-amide 1681 RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1682 GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1683 GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1684 GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1685 GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1686 GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1687 GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1688 GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1689 GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1690 GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1691 GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1692 GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK-amide 1693 GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK-amide 1694 GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK-amide 1695 GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK-amide 1696 GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK-amide 1697 GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK-amide 1698 GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK-amide 1699 GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK-amide 1700 GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK-amide 1701 GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK-amide 1702 GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK-amide 1703 GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK-amide 1704 GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK-amide 1705 GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK-amide 1706 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK-amide 1707 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK-amide 1708 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK-amide 1709 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK-amide 1710 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR-amide 1711 EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1712 GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1713 GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1714 GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1715 GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1716 GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1717 GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1718 GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1719 GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1720 GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1721 GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1722 GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK-amide 1723 GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK-amide 1724 GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK-amide 1725 GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK-amide 1726 GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK-amide 1727 GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK-amide 1728 GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK-amide 1729 GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK-amide 1730 GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK-amide 1731 GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK-amide 1732 GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK-amide 1733 GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK-amide 1734 GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK-amide 1735 GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK-amide 1736 GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK-amide 1737 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK-amide 1738 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK-amide 1739 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK-amide 1740 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK-amide 1741 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE-amide 1742 [1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1743 G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1744 GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1745 GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1746 GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1747 GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1748 GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1749 GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1750 GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1751 GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1752 GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1753 GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHCTPK- amide 1754 GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHCTPK- amide 1755 GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHCTPK- amide 1756 GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHCTPK- amide 1757 GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHCTPK- amide 1758 GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHCTPK- amide 1759 GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHCTPK- amide 1760 GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHCTPK- amide 1761 GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHCTPK- amide 1762 GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHCTPK- amide 1763 GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHCTPK- amide 1764 GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHCTPK- amide 1765 GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHCTPK- amide 1766 GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHCTPK- amide 1767 GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHCTPK- amide 1768 GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHCTPK- amide 1769 GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHCTPK- amide 1770 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]CTPK- amide 1771 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]PK- amide 1772 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-Nal]K- amide 1773 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-Nal]- amide 1774 Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1775 Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1776 Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1777 Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1778 Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1779 Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1780 Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1781 Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1782 Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1783 Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1784 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1785 Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK- amide 1786 Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK- amide 1787 Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK- amide 1788 Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK- amide 1789 Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK- amide 1790 Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK- amide 1791 Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK- amide 1792 Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK- amide 1793 Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK- amide 1794 Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK- amide 1795 Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK- amide 1796 Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- amide 1797 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- amide 1798 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- amide 1799 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK- amide 1800 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK- amide 1801 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK- amide 1802 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK- amide 1803 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK- amide 1804 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA- amide 1805 Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1806 Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1807 Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1808 Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1809 Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1810 Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1811 Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1812 Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1813 Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1814 Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1815 Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1816 Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK- amide 1817 Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK- amide 1818 Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK- amide 1819 Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK- amide 1820 Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK- amide 1821 Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK- amide 1822 Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK- amide 1823 Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK- amide 1824 Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK- amide 1825 Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK- amide 1826 Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK- amide 1827 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK- amide 1828 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK- amide 1829 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK- amide 1830 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK- amide 1831 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK- amide 1832 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK- amide 1833 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK- amide 1834 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR- amide 1835 Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1836 Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1837 Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1838 Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1839 Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1840 Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1841 Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1842 Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1843 Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1844 Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1845 Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK- amide 1846 Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK- amide 1847 Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK- amide 1848 Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK- amide 1849 Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK- amide 1850 Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK- amide 1851 Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK- amide 1852 Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK- amide 1853 Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK- amide 1854 Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK- amide 1855 Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK- amide 1856 Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK- amide 1857 Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK- amide 1858 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK- amide 1859 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK- amide 1860 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK- amide 1861 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK- amide 1862 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK- amide 1863 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK- amide 1864 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK- amide 1865 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE- amide 1866 Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide amide 1867 Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1868 Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1869 Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1870 Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1871 Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1872 Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1873 Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1874 Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1875 Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1876 Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKC HCTPK-amide 1877 Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKC HCTPK-amide 1878 Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKC HCTPK-amide 1879 Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKC HCTPK-amide 1880 Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKC HCTPK-amide 1881 Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKC HCTPK-amide 1882 Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKC HCTPK-amide 1883 Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKC HCTPK-amide 1884 Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKC HCTPK-amide 1885 Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKC HCTPK-amide 1886 Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKC HCTPK-amide 1887 Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKC HCTPK-amide 1888 Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKC HCTPK-amide 1889 Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKC HCTPK-amide 1890 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKC HCTPK-amide 1891 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKC HCTPK-amide 1892 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KC HCTPK-amide 1893 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal] CHCTPK-amide 1894 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC [1-Nal]CTPK-amide 1895 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [1-Nal]PK-amide 1896 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT [1-Nal]K-amide 1897 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP [1-Nal]-amide

TABLE 7B Additional useful OSK1 peptide analog sequences: Ala-12 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1898 GVIINVSCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1899 GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK 1900 GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK 1901 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1902 GVIINVSCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1903 GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK 1904 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK 1905 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK 1906 GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1907 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK- 1908 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK-amide 1909 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK 1910 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK- 1911 amide GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK-amide 1912 Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK 1913 Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK- 1914 amide Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK 1915 GVIINVKCKISAQCLEPCKDAGMRFGKCMNCKCHCTPK-amide 1916 Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK- 1917 amide GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1918 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1919 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- 1920 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1921 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1922 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1923 amide VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1924 Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1925 VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1926 Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- 1927 amide GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1928 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1929 GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide 1930 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK- 1931 amide VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1932 Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1933 VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1934 Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1935 amide NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1936 Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1937 NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1938 Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1939 KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1940 Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1941 KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1942 Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1943 CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1944 Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1945 CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1946 Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1947 GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK 1948 GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK-amide 1949 Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK 1950 Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK- 1951 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 1952 GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 1953 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 1954 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 1955 amide GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK 1956 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK 1957 GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-amide 1958 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK- 1959 amide TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1960 Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1961 TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1962 Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1963 amide GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1964 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK 1965 GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide 1966 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK- 1967 amide GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1968 GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1969 GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK 1970 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK 1971 GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide 1972 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK- 1973 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK 1974 GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK-amide 1975 Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK- 1976 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK 1977 GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide 1978 Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK- 1979 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCT 1980 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK 1981 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK 1982 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK 1983 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK 1984 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK 1985 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK 1986 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK 1987 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK 1988 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK 1989 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK 1990 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK 1991 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK 1992 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK 1993 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK 1994 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYPK 1995 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYPK 1996 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFPK 1997 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACWPK 1998 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYPK 1999 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK 2000 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK 2001 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK 2002 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK 2003 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK 2004 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK 2005 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK 2006 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC 2007 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC 2008 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC 2009 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC 2010 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC 2011 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC 2012 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHC 2013 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC 2014 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC 2015 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC 2016 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC 2017 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC 2018 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC 2019 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC 2020 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYGG 2021 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG 2022 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG 2023 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG 2024 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG 2025 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG 2026 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG 2027 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG 2028 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG 2029 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG 2030 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG 2031 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG 2032 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG 2033 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG 2034 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG 2035 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG 2036 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG 2037 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG 2038 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG 2039 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG 2040 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG 2041 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG 2042 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFGG 2043 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG 2044 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG 2045 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG 2046 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG 2047 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG 2048 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG 2049 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG 2050 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGG 2051 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG 2052 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGG 2053 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK 2054 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK 2055 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK 2056 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK 2057 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK 2058 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK 2059 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK 2060 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK 2061 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK 2062 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK 2063 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK 2064 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK 2065 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK 2066 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK 2067 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK 2068 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK 2069 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK 2070 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK 2071 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK 2072 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK 2073 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC 2074 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC 2075 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC 2076 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC 2077 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC 2078 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC 2079 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC 2080 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC 2081 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC 2082 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC 2083 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC 2084 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC 2085 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC 2086 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYGG 2087 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG 2088 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYG 2089 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG 2090 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG 2091 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG 2092 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG 2093 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG 2094 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG 2095 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG 2096 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG 2097 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG 2098 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG 2099 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG 2100 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG 2101 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG 2102 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG 2103 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG 2104 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACFGG 2105 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG 2106 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG 2107 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG 2108 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG 2109 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG 2110 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG 2111 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG 2112 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG 2113 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG 2114 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG 2115 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP 2116 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTP 2117 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTP 2118 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK-amide 2119 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK- 2120 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK- 2121 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK- 2122 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK- 2123 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK- 2124 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK- 2125 amide GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK-amide 2126 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK- 2127 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK- 2128 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK- 2129 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK- 2130 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK- 2131 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK- 2132 amide GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK-amide 2133 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK- 2134 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK- 2135 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK- 2136 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACTPK- 2137 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK- 2138 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK- 2139 amide GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC-amide 2140 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC- 2141 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC- 2142 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC-amide 2143 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC- 2144 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC-amide 2145 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC-amide 2146 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC- 2147 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC- 2148 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC-amide 2149 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC- 2150 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC-amide 2151 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC-amide 2152 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYGG-amide 2153 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG-amide 2154 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG- 2155 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG- 2156 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG- 2157 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG- 2158 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG- 2159 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCFGG-amide 2160 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYG-amide 2161 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG-amide 2162 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG-amide 2163 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG- 2164 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG- 2165 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG- 2166 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACYGG- 2167 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG- 2168 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG- 2169 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG-amide 2170 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG-amide 2171 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG- 2172 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG- 2173 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG- 2174 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG- 2175 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG- 2176 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGGG-amide 2177 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACFGG-amide 2178 GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG-amide 2179 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG- 2180 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG- 2181 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG- 2182 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG- 2183 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG- 2184 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG- 2185 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCTPK-amide 2186 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK- 2187 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK- 2188 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK- 2189 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK- 2190 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK- 2191 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK- 2192 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK-amide 2193 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK- 2194 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK- 2195 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK- 2196 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK- 2197 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK- 2198 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK- 2199 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK-amide 2200 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK- 2201 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK- 2202 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK- 2203 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTPK- 2204 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK- 2205 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK- 2206 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC-amide 2207 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC- 2208 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC- 2209 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC-amide 2210 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC- 2211 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC-amide 2212 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC-amide 2213 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC- 2214 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC- 2215 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC-amide 2216 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC- 2217 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC-amide 2218 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC-amide 2219 GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCWGG-amide 2220 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG-amide 2221 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG- 2222 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG- 2223 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG- 2224 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- 2225 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG- 2226 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG- 2227 amide GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYGG-amide 2228 GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG-amide 2229 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG- 2230 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG- 2231 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG- 2232 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG- 2233 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG- 2234 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG- 2235 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG-amide 2236 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG- 2237 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG- 2238 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG- 2239 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG- 2240 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG- 2241 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG-amide 2242 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG- 2243 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG- 2244 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG- 2245 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP- 2246 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG- 2247 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACGGG- 2248 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG- 2249 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG- 2250 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACY- 2251 amide Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG- 2252 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG- 2253 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG- 2254 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG 2255 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG-amide 2256 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG 2257 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACY-amide 2258 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG-amide 2259 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG 2260 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYPK 2261 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC 2262 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG- 2263 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK- 2264 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG- 2265 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG- 2266 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[MePhe]GG- 2267 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]GG- 2268 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG- 2269 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG- 2270 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal] 2271 GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG- 2272 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[4Bip]- 2273 amide GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG- 2274 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCGGG 2275

TABLE 7C Additional useful OSK1 peptide analog sequences: Ala-12 & Ala27 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2276 GVIINVSCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2277 GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK 2278 GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK 2279 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2280 GVIINVSCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2281 GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 2282 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK 2283 Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 2284 GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2285 Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK- 2286 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK-amide 2287 Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK 2288 Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK- 2289 amide GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK-amide 2290 Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK 2291 Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK- 2292 amide Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK 2293 GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK-amide 2294 Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK- 2295 amide GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide 2296 Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2297 Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK- 2298 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2299 Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2300 Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2301 amide VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2302 Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2303 VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide 2304 Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK- 2305 amide GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2306 Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2307 GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide 2308 Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK- 2309 amide VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2310 Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2311 VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2312 Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2313 amide NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2314 Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2315 NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2316 Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2317 KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2318 Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2319 KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2320 Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2321 CKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2322 Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2323 CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2324 Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2325 GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK 2326 GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK-amide 2327 Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK 2328 Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK- 2329 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2330 GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 2331 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2332 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 2333 amide GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 2334 Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 2335 GVIINVKCKISKQCLKPCGDAGMRFGACMNGKCHCTPK-amide 2336 Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK- 2337 amide TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2338 Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2339 TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2340 Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2341 amide GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2342 Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK 2343 GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide 2344 Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK- 2345 amide GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2346 GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2347 GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK 2348 Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK 2349 GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide 2350 Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK- 2351 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK 2352 GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK-amide 2353 Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK- 2354 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK 2355 GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide 2356 Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK- 2357 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCT 2358 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK 2359 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK 2360 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK 2361 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK 2362 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 2363 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK 2364 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK 2365 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK 2366 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK 2367 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK 2368 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK 2369 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK 2370 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK 2371 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK 2372 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYPK 2373 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYPK 2374 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFPK 2375 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACWPK 2376 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYPK 2377 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK 2378 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK 2379 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK 2380 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK 2381 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 2382 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK 2383 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK 2384 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC 2385 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC 2386 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC 2387 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC 2388 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC 2389 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC 2390 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHC 2391 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC 2392 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC 2393 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC 2394 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC 2395 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC 2396 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC 2397 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC 2398 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYGG 2399 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG 2400 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG 2401 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG 2402 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG 2403 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 2404 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG 2405 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG 2406 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG 2407 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG 2408 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG 2409 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG 2410 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 2411 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG 2412 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG 2413 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG 2415 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG 2416 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG 2417 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG 2418 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG 2419 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG 2420 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG 2421 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFGG 2422 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG 2423 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG 2424 GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG 2425 GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG 2426 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG 2427 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG 2428 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG 2429 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGG 2430 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG 2431 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGG 2432 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK 2433 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK 2434 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK 2435 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 2436 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK 2437 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK 2438 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK 2439 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK 2440 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK 2441 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK 2442 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK 2443 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK 2444 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK 2445 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK 2446 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK 2447 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK 2448 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK 2449 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 2450 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK 2451 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK 2452 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC 2453 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC 2454 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC 2455 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC 2456 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC 2457 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC 2458 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC 2459 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC 2460 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC 2461 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC 2462 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC 2463 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC 2464 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC 2465 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYGG 2466 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG 2467 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYG 2468 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG 2469 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG 2470 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG 2471 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG 2472 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 2473 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG 2474 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG 2475 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG 2476 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG 2477 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG 2478 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG 2479 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG 2480 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 2481 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG 2482 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG 2483 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACFGG 2484 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG 2485 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG 2486 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG 2487 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG 2488 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG 2489 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG 2490 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG 2491 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG 2492 GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG 2493 GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG 2494 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP 2495 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTP 2496 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTP 2497 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK-amide 2498 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK- 2499 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK- 2500 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK- 2501 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK- 2502 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK- 2503 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK- 2504 amide GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK-amide 2505 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK- 2506 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK- 2507 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK- 2508 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK- 2509 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK- 2510 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK- 2511 amide GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK-amide 2512 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK- 2513 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK- 2514 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK- 2515 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACTPK- 2516 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK- 2517 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK- 2518 amide GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC-amide 2519 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC- 2520 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC- 2521 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC- 2522 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC- 2523 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide 2524 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC-amide 2525 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC- 2526 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC- 2527 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC-amide 2528 GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC- 2529 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC-amide 2530 GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC-amide 2531 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYGG-amide 2532 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG-amide 2533 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG- 2534 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG- 2535 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG- 2536 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG- 2537 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG- 2538 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCFGG-amide 2539 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYG-amide 2540 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG-amide 2541 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG-amide 2542 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG- 2543 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG- 2544 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG- 2545 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACYGG- 2546 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG- 2547 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG- 2548 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG-amide 2549 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG-amide 2550 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG- 2551 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG- 2552 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG- 2553 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG- 2554 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG- 2555 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGGG-amide 2556 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACFGG-amide 2557 GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG-amide 2558 GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG- 2559 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG- 2560 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG- 2561 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG- 2562 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG- 2563 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG- 2564 amide GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCTPK-amide 2565 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK- 2566 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK- 2567 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK- 2568 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK- 2569 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK- 2570 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK- 2571 amide GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK-amide 2572 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK- 2573 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK- 2574 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK- 2575 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK- 2576 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK- 2577 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK- 2578 amide GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK-amide 2579 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK- 2580 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK- 2581 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK- 2582 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTPK- 2583 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK- 2584 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK- 2585 amide GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC-amide 2586 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC- 2587 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC- 2588 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC-amide 2589 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC- 2590 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC-amide 2591 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC-amide 2592 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC- 2593 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC- 2594 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC-amide 2595 GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC- 2596 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC-amide 2597 GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC-amide 2598 GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCWGG-amide 2599 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG-amide 2600 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG- 2601 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG- 2602 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG- 2603 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 2604 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG- 2605 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG- 2606 amide GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYGG-amide 2607 GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG-amide 2608 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG- 2609 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG- 2610 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG- 2611 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 2612 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG- 2613 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG- 2614 amide GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG-amide 2615 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG- 2616 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG- 2617 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG- 2618 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG- 2619 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG- 2620 amide GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG-amide 2621 GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG- 2622 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG- 2623 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG- 2624 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP- 2625 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACGGG- 2626 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACGGG- 2627 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACYGG- 2628 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACGGG- 2629 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACY- 2630 amide Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG- 2631 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG- 2632 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCHCYGG- 2633 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG 2634 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG-amide 2635 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG 2636 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACY-amide 2637 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG-amide 2638 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG 2639 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYPK 2640 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC 2641 GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG- 2642 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK- 2643 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG- 2644 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[Cha]GG- 2645 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG- 2646 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG- 2647 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Aib]CYGG- 2648 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Abu]CYGG- 2649 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal] 2650 GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG- 2651 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[4Bip]- 2652 amide GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG- 2653 amide GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCGGG 2654

TABLE 7D Additional useful OSK1 peptide analog sequences: Ala-12 & Ala29 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2655 GVIINVSCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2656 GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK 2657 GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK 2658 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2659 GVIINVSCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2660 GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 2661 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK 2662 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 2663 GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2664 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK- 2665 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK-amide 2666 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK 2667 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK- 2668 amide GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK-amide 2669 Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK 2670 Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK- 2671 amide Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK 2672 GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK-amide 2673 Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK- 2674 amide GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide 2675 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2676 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK- 2677 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2678 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2679 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2680 amide VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2681 Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2682 VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide 2683 Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK- 2684 amide GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2685 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2686 GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide 2687 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK- 2688 amide VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2689 Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2690 VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2691 Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2692 amide NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2693 Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2694 NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2695 Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2696 KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2697 Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2698 KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2699 Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2700 CKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2701 Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2702 CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2703 Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2704 GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK 2705 GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK-amide 2706 Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK 2707 Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK- 2708 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2709 GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 2710 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 2711 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 2712 amide GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 2713 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 2714 GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide 2715 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK- 2716 amide TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2717 Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2718 TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2719 Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2720 amide GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2721 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK 2722 GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide 2723 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK- 2724 amide GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2725 GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2726 GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK 2727 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK 2728 GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide 2729 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK- 2730 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK 2731 GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK-amide 2732 Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK- 2733 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK 2734 GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide 2735 Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK- 2736 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCT 2737 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK 2738 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK 2739 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK 2740 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK 2741 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 2742 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK 2743 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK 2744 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK 2745 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK 2746 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK 2747 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK 2748 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK 2749 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK 2750 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK 2751 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYPK 2752 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYPK 2753 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFPK 2754 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACWPK 2755 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYPK 2756 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK 2757 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK 2758 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK 2759 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK 2760 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 2761 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK 2762 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK 2763 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC 2764 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC 2765 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC 2766 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC 2767 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC 2768 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC 2769 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHC 2770 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC 2771 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC 2772 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC 2773 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC 2774 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC 2775 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC 2776 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC 2777 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYGG 2778 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG 2779 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG 2780 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG 2781 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG 2782 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 2783 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG 2784 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG 2785 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG 2786 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG 2787 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG 2788 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG 2789 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 2790 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG 2791 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG 2792 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG 2793 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG 2794 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG 2795 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG 2796 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG 2797 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG 2798 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG 2799 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFGG 2800 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG 2801 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG 2802 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG 2803 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG 2804 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG 2805 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG 2806 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG 2807 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGG 2808 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG 2809 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGG 2810 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK 2811 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK 2812 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK 2813 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 2814 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK 2815 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK 2816 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK 2817 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK 2818 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK 2819 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK 2820 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK 2821 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK 2822 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK 2823 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK 2824 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK 2825 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK 2826 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK 2827 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 2828 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK 2829 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK 2830 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC 2831 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC 2832 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC 2833 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC 2834 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC 2835 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC 2836 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC 2837 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC 2838 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC 2839 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC 2840 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC 2841 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC 2842 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC 2843 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYGG 2844 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG 2845 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYG 2846 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG 2847 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG 2848 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG 2849 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG 2850 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 2851 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG 2852 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG 2853 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG 2854 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG 2855 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG 2856 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG 2857 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG 2858 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 2859 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG 2860 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG 2861 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACFGG 2862 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG 2863 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG 2864 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG 2865 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG 2866 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG 2867 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG 2868 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG 2869 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG 2870 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG 2871 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG 2872 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP 2873 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTP 2874 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTP 2875 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK-amide 2876 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK- 2877 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK- 2878 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK- 2879 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK- 2880 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK- 2881 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK- 2882 amide GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK-amide 2883 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK- 2884 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK- 2885 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK- 2886 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK- 2887 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK- 2888 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK- 2889 amide GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK-amide 2890 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK- 2891 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK- 2892 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK- 2893 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACTPK- 2894 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK- 2895 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK- 2896 amide GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC-amide 2897 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC- 2898 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC- 2899 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC-amide 2900 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC- 2901 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide 2902 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC-amide 2903 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC- 2904 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC- 2905 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC-amide 2906 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC- 2907 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC-amide 2908 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC-amide 2909 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYGG-amide 2910 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG-amide 2911 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG- 2912 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG- 2913 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG- 2914 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG- 2915 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG- 2916 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCFGG-amide 2917 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYG-amide 2918 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG-amide 2919 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG-amide 2920 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG- 2921 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG- 2922 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG- 2923 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACYGG- 2924 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG- 2925 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG- 2926 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG-amide 2927 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG-amide 2928 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG- 2929 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG- 2930 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG- 2931 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG- 2932 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG- 2933 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGGG-amide 2934 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACFGG-amide 2935 GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG-amide 2936 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG- 2937 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG- 2938 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG- 2939 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG- 2940 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG- 2941 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG- 2942 amide GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCTPK-amide 2943 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK- 2944 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK- 2945 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK- 2946 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK- 2947 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK- 2948 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK- 2949 amide GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK-amide 2950 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK- 2951 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK- 2952 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK- 2953 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK- 2954 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK- 2955 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK- 2956 amide GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK-amide 2957 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK- 2958 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK- 2959 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK- 2960 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTPK- 2961 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK- 2962 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK- 2963 amide GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC-amide 2964 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC- 2965 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC- 2966 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC-amide 2967 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC- 2968 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide 2969 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC-amide 2970 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC- 2971 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC- 2972 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC-amide 2973 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC- 2974 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide 2975 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC-amide 2976 GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCWGG-amide 2977 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG-amide 2978 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG- 2979 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG- 2980 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG- 2981 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 2982 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG- 2983 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG- 2984 amide GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYGG-amide 2985 GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG-amide 2986 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG- 2987 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG- 2988 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG- 2989 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 2990 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG- 2991 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG- 2992 amide GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG-amide 2993 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG- 2994 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG- 2995 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG- 2996 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG- 2997 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG- 2998 amide GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG-amide 2999 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG- 3000 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG- 3001 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG- 3002 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP- 3003 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACGGG- 3004 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACGGG- 3005 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACYGG- 3006 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACGGG- 3007 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACY- 3008 amide Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG- 3009 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG- 3010 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCHCYGG- 3011 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG 3012 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG-amide 3013 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG 3014 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACY-amide 3015 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG-amide 3016 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG 3017 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYPK 3018 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC 3019 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG- 3020 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK- 3021 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG- 3022 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[Cha]GG- 3023 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG- 3024 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG- 3025 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Aib]CYGG- 3026 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Abu]CYGG- 3027 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal] 3028 GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG- 3029 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[4Bip]- 3030 amide GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG- 3031 amide GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCGGG 3032

TABLE 7E Additional useful OSK1 peptide analogs: Ala-12 & Ala30 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3033 GVIINVSCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3034 GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK 3035 GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK 3036 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3037 GVIINVSCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3038 GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 3039 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK 3040 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 3041 GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3042 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK- 3043 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK-amide 3044 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK 3045 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK- 3046 amide GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK-amide 3047 Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK 3048 Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK- 3049 amide Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK 3050 GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK-amide 3051 Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK- 3052 amide GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 3053 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3054 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK- 3055 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3056 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3057 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3058 amide VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3059 Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3060 VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 3061 Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK- 3062 amide GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3063 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3064 GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide 3065 Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK- 3066 amide VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3067 Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3068 VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3069 Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3070 amide NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3071 Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3072 NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3073 Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3074 KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3075 Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3076 KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3077 Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3078 CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3079 Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3080 CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3081 Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3082 GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK 3083 GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK-amide 3084 Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK 3085 Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK- 3086 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 3087 GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 3088 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK 3089 Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK- 3090 amide GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 3091 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 3092 GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide 3093 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK- 3094 amide TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3095 Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3096 TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3097 Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3098 amide GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3099 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK 3100 GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 3101 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK- 3102 amide GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3103 GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3104 GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK 3105 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK 3106 GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide 3107 Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK- 3108 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK 3109 GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK-amide 3110 Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK- 3111 amide Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK 3112 GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 3113 Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK- 3114 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCT 3115 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK 3116 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK 3117 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK 3118 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK 3119 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 3120 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK 3121 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK 3122 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK 3123 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK 3124 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK 3125 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK 3126 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK 3127 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK 3128 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK 3129 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYPK 3130 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYPK 3131 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFPK 3132 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACWPK 4920 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYPK 4921 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK 4922 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK 4923 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK 4924 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK 4925 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 4926 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK 4927 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK 4928 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC 4929 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC 3133 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHC 3134 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC 3135 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC 3136 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC 3137 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHC 3138 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC 3139 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC 3140 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC 3141 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC 3142 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC 3143 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC 3144 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC 3145 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYGG 3146 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG 3147 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG 3148 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG 3149 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG 3150 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 3151 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG 3152 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG 3153 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG 3154 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG 3155 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG 3156 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG 3157 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 3158 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG 3159 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG 3160 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG 3161 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG 3162 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG 3163 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG 3164 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG 3165 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG 3166 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG 3167 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFGG 3168 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG 3169 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG 3170 GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG 3171 GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG 3172 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG 3173 GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG 3174 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG 3175 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGG 3176 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG 3177 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGG 3178 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK 3179 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK 3180 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK 3181 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 3182 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK 3183 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK 3184 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK 3185 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK 3186 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK 3187 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK 3188 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK 3189 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK 3190 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK 3191 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK 3192 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK 3193 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK 3194 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK 3195 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 3196 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK 3197 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK 3198 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC 3199 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC 3200 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC 3201 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC 3202 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC 3203 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC 3204 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC 3205 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC 3206 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC 3207 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC 3208 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC 3209 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC 3210 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC 3211 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYGG 3212 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG 3213 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYG 3214 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG 3215 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG 3216 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG 3217 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG 3218 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 3219 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG 3220 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG 3221 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG 3222 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG 3223 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG 3224 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG 3225 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG 3226 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 3227 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG 3228 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG 3229 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACFGG 3230 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG 3231 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG 3232 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG 3233 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG 3234 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG 3235 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG 3236 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG 3237 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG 3238 GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG 3239 GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG 3240 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP 3241 GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTP 3242 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTP 3243 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK-amide 3244 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK- 3245 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK- 3246 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK- 3247 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK- 3248 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK- 3249 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK- 3250 amide GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK-amide 3251 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK- 3252 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK- 3253 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK- 3254 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK- 3255 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK- 3256 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK- 3257 amide GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK-amide 3258 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK- 3259 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK- 3260 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK- 3261 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACTPK- 3262 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK- 3263 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK- 3264 amide GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC-amide 3265 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC- 3266 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHC- 3267 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide 3268 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC- 3269 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide 3270 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC-amide 3271 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC- 3272 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC- 3273 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide 3274 GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC- 3275 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC-amide 3276 GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide 3277 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYGG-amide 3278 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG-amide 3279 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG- 3280 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG- 3281 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG- 3282 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG- 3283 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG- 3284 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCFGG-amide 3285 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYG-amide 3286 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG-amide 3287 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG-amide 3288 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG- 3289 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG- 3290 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG- 3291 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACYGG- 3292 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG- 3293 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG- 3294 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG-amide 3295 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG-amide 3296 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG- 3297 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG- 3298 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG- 3299 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG- 3300 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG- 3301 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGGG-amide 3302 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACFGG-amide 3303 GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG-amide 3304 GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG- 3305 amide GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG- 3306 amide GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG- 3307 amide GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG- 3308 amide GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG- 3309 amide GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG- 3310 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCTPK-amide 3311 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK- 3312 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK- 3313 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK- 3314 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK- 3315 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK- 3316 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK- 3317 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK-amide 3318 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK- 3319 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK- 3320 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK- 3321 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK- 3322 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK- 3323 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK- 3324 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK-amide 3325 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK- 3326 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK- 3327 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK- 3328 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTPK- 3329 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK- 3330 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK- 3331 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC-amide 3332 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC- 3333 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC- 3334 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide 3335 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC- 3336 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide 3337 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC-amide 3338 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC- 3339 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC- 3340 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide 3341 GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC- 3342 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide 3343 GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide 3344 GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCWGG-amide 3345 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG-amide 3346 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG- 3347 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG- 3348 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG- 3349 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 3350 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG- 3351 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG- 3352 amide GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYGG-amide 3353 GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG-amide 3354 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG- 3355 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG- 3356 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG- 3357 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 3358 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG- 3359 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG- 3360 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG-amide 3361 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG- 3362 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG- 3363 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG- 3364 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG- 3365 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG- 3366 amide GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG-amide 3367 GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG- 3368 amide GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG- 3369 amide GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG- 3370 amide GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP- 3371 amide GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG- 3372 amide GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACGGG- 3373 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG- 3374 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG- 3375 amide GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACY- 3376 amide Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG- 3377 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG- 3378 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG- 3379 amide GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG 3380 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG-amide 3381 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG 3382 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACY-amide 3383 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG-amide 3384 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG 3385 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYPK 3386 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC 3387 GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG- 3388 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK- 3389 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG- 3390 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG- 3391 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG- 3392 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG- 3393 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG- 3394 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG- 3395 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal] 3396 GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG- 3397 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[4Bip]- 3398 amide GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG- 3399 amide GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCGGG 3400

TABLE 7F Addit6ional useful OSK1 peptide analogs: Ala27Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3401 GVIINVSCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3402 GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK 3403 GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK 3404 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3405 GVIINVSCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3406 GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 3407 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK 3408 Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK 3409 GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3410 Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK- 3411 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK-amide 3412 Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK 3413 Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK- 3414 amide GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK-amide 3415 Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK 3416 Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK- 3417 amide Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK 3418 GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK-amide 3419 Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK- 3420 amide GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 3421 Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3422 Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- 3423 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3424 Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3425 Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3426 amide VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3427 Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3428 VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 3429 Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- 3430 amide GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3431 Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3432 GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide 3433 Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK- 3434 amide VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3435 Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3436 VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3437 Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3438 amide NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3439 Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3440 NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3441 Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3442 KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3443 Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3444 KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3445 Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3446 CKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3447 Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3448 CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3449 Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3450 GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK 3451 GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK-amide 3452 Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK 3453 Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK- 3454 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3455 GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 3456 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3457 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK- 3458 amide GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 3459 Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK 3460 GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-amide 3461 Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK- 3462 amide TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3463 Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3464 TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3465 Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3466 amide GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3467 Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK 3468 GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide 3469 Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK- 3470 amide GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3471 GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3472 GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK 3473 Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK 3474 GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide 3475 Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK- 3476 amide Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK 3477 GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK-amide 3478 Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK- 3479 amide Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK 3480 GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide 3481 Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK- 3482 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCT 3483 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK 3484 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK 3485 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK 3486 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK 3487 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 3488 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK 3489 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK 3490 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK 3491 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK 3492 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK 3493 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK 3494 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK 3495 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK 3496 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK 3497 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYPK 3498 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYPK 3499 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFPK 3500 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACWPK 3501 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYPK 3502 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK 3503 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK 3504 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK 3505 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK 3506 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK 3507 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK 3508 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK 3509 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC 3510 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC 3511 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC 3512 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC 3513 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC 3514 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC 3515 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHC 3516 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC 3517 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC 3518 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC 3519 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC 3520 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC 3521 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC 3522 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC 3523 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYGG 3524 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG 3525 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG 3526 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG 3527 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG 3528 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 3529 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG 3530 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG 3531 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG 3532 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG 3533 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG 3534 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG 3535 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG 3536 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG 3537 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG 3538 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG 3539 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG 3540 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG 3541 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG 3542 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG 3543 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG 3544 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG 3545 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFGG 3546 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG 3547 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG 3548 GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG 3549 GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG 3550 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG 3551 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG 3552 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG 3553 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGG 3554 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG 3555 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGG 3556 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK 3557 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK 3558 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK 3559 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 3560 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK 3561 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK 3562 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK 3563 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK 3564 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK 3565 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK 3566 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK 3567 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK 3568 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK 3569 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK 3570 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK 3571 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK 3572 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK 3573 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK 3574 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK 3575 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK 3576 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC 3577 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC 3578 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC 3579 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC 3580 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC 3581 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC 3582 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC 3583 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC 3584 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC 3585 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC 3586 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC 3587 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC 3588 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC 3589 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYGG 3590 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG 3591 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYG 3592 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG 3593 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG 3594 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG 3595 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG 3596 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 3597 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG 3598 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG 3599 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG 3600 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG 3601 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG 3602 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG 3603 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG 3604 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG 3605 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG 3606 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG 3607 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACFGG 3608 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG 3609 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG 3610 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG 3611 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG 3612 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG 3613 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG 3614 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG 3615 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG 3616 GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG 3617 GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG 3618 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP 3619 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTP 3620 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTP 3621 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK-amide 3622 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK- 3623 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK- 3624 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK- 3625 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK- 3626 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK- 3627 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK- 3628 amide GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK-amide 3629 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK- 3630 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK- 3631 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK- 3632 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK- 3633 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK- 3634 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK- 3635 amide GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK-amide 3636 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK- 3637 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK- 3638 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK- 3639 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACTPK- 3640 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK- 3641 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK- 3642 amide GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC-amide 3643 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC- 3644 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC- 3645 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC-amide 3646 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC- 3647 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide 3648 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC-amide 3649 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC- 3650 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC- 3651 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC-amide 3652 GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC- 3653 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC-amide 3654 GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC-amide 3655 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYGG-amide 3656 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG-amide 3657 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG- 3658 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG- 3659 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG- 3660 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG- 3661 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG- 3662 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCFGG-amide 3663 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYG-amide 3664 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG-amide 3665 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG-amide 3666 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG- 3667 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG- 3668 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG- 3669 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACYGG- 3670 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG- 3671 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG- 3672 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG-amide 3673 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG-amide 3674 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG- 3675 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG- 3676 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG- 3677 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG- 3678 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG- 3679 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGGG-amide 3680 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACFGG-amide 3681 GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG-amide 3682 GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG- 3683 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG- 3684 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG- 3685 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG- 3686 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG- 3687 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG- 3688 amide GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCTPK-amide 3689 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK- 3690 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK- 3691 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK- 3692 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK- 3693 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK- 3694 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK- 3695 amide GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK-amide 3696 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK- 3697 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK- 3698 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK- 3699 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK- 3700 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK- 3701 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK- 3702 amide GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK-amide 3703 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK- 3704 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK- 3705 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK- 3706 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTPK- 3707 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK- 3708 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK- 3709 amide GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC-amide 3710 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC- 3711 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC- 3712 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC-amide 3713 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC- 3714 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC-amide 3715 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC-amide 3716 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC- 3717 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC- 3718 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC-amide 3719 GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC- 3720 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC-amide 3721 GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC-amide 3722 GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCWGG-amide 3723 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG-amide 3724 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG- 3725 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG- 3726 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG- 3727 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 3728 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG- 3729 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG- 3730 amide GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYGG-amide 3731 GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG-amide 3732 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG- 3733 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG- 3734 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG- 3735 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG- 3736 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG- 3737 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG- 3738 amide GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG-amide 3739 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG- 3740 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG- 3741 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG- 3742 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG- 3743 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG- 3744 amide GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG-amide 3745 GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG- 3746 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG- 3747 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG- 3748 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP- 3749 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACGGG- 3750 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACGGG- 3751 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACYGG- 3752 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACGGG- 3753 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACY- 3754 amide Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG- 3755 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG- 3756 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCHCYGG- 3757 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG 3758 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG-amide 3759 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG 3760 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACY-amide 3761 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG-amide 3762 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG 3763 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYPK 3764 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC 3765 GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG- 3766 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK- 3767 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG- 3768 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[Cha]GG- 3769 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG- 3770 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG- 3771 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Aib]CYGG- 3772 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Abu]CYGG- 3773 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal] 3774 GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG- 3775 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[4Bip]- 3776 amide GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG- 3777 amide GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCGGG 3778

TABLE 7G Additional useful OSK1 peptide analogs: Ala 29 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3779 GVIINVSCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3780 GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK 3781 GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK 3782 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3783 GVIINVSCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3784 GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 3785 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK 3786 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK 3787 GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3788 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK- 3789 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK-amide 3790 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK 3791 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK- 3792 amide GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK-amide 3793 Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK 3794 Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK- 3795 amide Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK 3796 GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK-amide 3797 Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK- 3798 amide GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 3799 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3800 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- 3801 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3802 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3803 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3804 amide VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3805 Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3806 VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 3807 Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- 3808 amide GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3809 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3810 GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide 3811 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK- 3812 amide VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3813 Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3814 VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3815 Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3816 amide NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3817 Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3818 NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3819 Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3820 KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3821 Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3822 KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3823 Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3824 CKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3825 Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3826 CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3827 Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3828 GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK 3829 GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK-amide 3830 Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK 3831 Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK- 3832 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3833 GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 3834 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 3835 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK- 3836 amide GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 3837 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK 3838 GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide 3839 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK- 3840 amide TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3841 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3842 TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3843 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3844 amide GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3845 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK 3846 GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide 3847 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK- 3848 amide GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3849 GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3850 GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK 3851 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK 3852 GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide 3853 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK- 3854 amide Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK 3855 GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK-amide 3856 Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK- 3857 amide Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK 3858 GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide 3859 Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK- 3860 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCT 3861 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK 3862 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK 3863 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK 3864 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK 3865 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 3866 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK 3867 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK 3868 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK 3869 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK 3870 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK 3871 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK 3872 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK 3873 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK 3874 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK 3875 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYPK 3876 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYPK 3877 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFPK 3878 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACWPK 3879 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYPK 3880 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK 3881 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK 3882 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK 3883 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK 3884 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK 3885 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK 3886 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK 3887 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC 3888 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC 3889 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC 3890 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC 3891 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC 3892 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC 3893 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHC 3894 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC 3895 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC 3896 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC 3897 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC 3898 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC 3899 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC 3900 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC 3901 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYGG 3902 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG 3903 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG 3904 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG 3905 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG 3906 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 3907 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG 3908 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG 3909 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG 3910 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG 3911 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG 3912 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG 3913 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG 3914 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG 3915 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG 3916 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG 3917 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG 3918 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG 3919 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG 3920 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG 3921 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG 3922 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG 3923 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFGG 3924 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG 3925 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG 3926 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG 3927 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG 3928 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG 3929 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG 3930 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG 3931 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGG 3932 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG 3933 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGG 3934 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK 3935 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK 3936 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK 3937 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 3938 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK 3939 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK 3940 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK 3941 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK 3942 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK 3943 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK 3944 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK 3945 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK 3946 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK 3947 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK 3948 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK 3949 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK 3950 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK 3951 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK 3952 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK 3953 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK 3954 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC 3955 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC 3956 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC 3957 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC 3958 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC 3959 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC 3960 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC 3961 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC 3962 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC 3963 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC 3964 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC 3965 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC 3966 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC 3967 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYGG 3968 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG 3969 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYG 3970 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG 3971 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG 3972 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG 3973 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG 3974 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 3975 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG 3976 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG 3977 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG 3978 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG 3979 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG 3980 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG 3981 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG 3982 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG 3983 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG 3984 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG 3985 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACFGG 3986 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG 3987 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG 3988 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG 3989 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG 3990 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG 3991 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG 3992 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG 3993 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG 3994 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG 3995 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG 3996 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP 3997 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTP 3998 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTP 3999 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK-amide 4000 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK- 4001 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK- 4002 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK- 4003 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK- 4004 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK- 4005 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK- 4006 amide GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK-amide 4007 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK- 4008 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK- 4009 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK- 4010 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK- 4011 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK- 4012 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK- 4013 amide GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK-amide 4014 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK- 4015 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK- 4016 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK- 4017 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACTPK- 4018 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK- 4019 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK- 4020 amide GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC-amide 4021 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC- 4022 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC- 4023 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC-amide 4024 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC- 4025 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide 4026 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC-amide 4027 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC- 4028 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC- 4029 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC-amide 4030 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC- 4031 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC-amide 4032 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC-amide 4033 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYGG-amide 4034 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG-amide 4035 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG- 4036 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG- 4037 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG- 4038 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG- 4039 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG- 4040 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCFGG-amide 4041 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYG-amide 4042 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG-amide 4043 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG-amide 4044 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG- 4045 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG- 4046 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG- 4047 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACYGG- 4048 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG- 4049 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG- 4050 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG-amide 4051 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG-amide 4052 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG- 4053 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG- 4054 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG- 4055 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG- 4056 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG- 4057 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGGG-amide 4058 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACFGG-amide 4059 GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG-amide 4060 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG- 4061 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG- 4062 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG- 4063 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG- 4064 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG- 4065 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG- 4066 amide GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCTPK-amide 4067 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK- 4068 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK- 4069 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK- 4070 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK- 4071 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK- 4072 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK- 4073 amide GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK-amide 4074 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK- 4075 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK- 4076 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK- 4077 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK- 4078 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK- 4079 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK- 4080 amide GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK-amide 4081 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK- 4082 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK- 4083 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK- 4084 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTPK- 4085 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK- 4086 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK- 4087 amide GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC-amide 4088 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC- 4089 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC- 4090 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC-amide 4091 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC- 4092 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide 4093 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC-amide 4094 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC- 4095 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC- 4096 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC-amide 4097 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC- 4098 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide 4099 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC-amide 4100 GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCWGG-amide 4101 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG-amide 4102 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG- 4103 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG- 4104 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG- 4105 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 4106 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG- 4107 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG- 4108 amide GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYGG-amide 4109 GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG-amide 4110 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG- 4111 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG- 4112 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG- 4113 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG- 4114 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG- 4115 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG- 4116 amide GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG-amide 4117 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG- 4118 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG- 4119 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG- 4120 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG- 4121 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG- 4122 amide GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG-amide 4123 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG- 4124 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG- 4125 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG- 4126 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP- 4127 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACGGG- 4128 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACGGG- 4129 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACYGG- 4130 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACGGG- 4131 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACY- 4132 amide Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG- 4133 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG- 4134 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCHCYGG- 4135 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG 4136 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG-amide 4137 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG 4138 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACY-amide 4139 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG-amide 4140 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG 4141 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYPK 4142 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC 4143 GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG- 4144 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK- 4145 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG- 4146 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[Cha]GG- 4147 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG- 4148 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG- 4149 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Aib]CYGG- 4150 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Abu]CYGG- 4151 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal] 4152 GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG- 4153 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[4Bip]- 4154 amide GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG- 4155 amide GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCGGG 4156

TABLE 7H Additional useful OSK1 peptide analogs: Ala 30 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4157 GVIINVSCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4158 GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK 4159 GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK 4160 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4161 GVIINVSCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4162 GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 4163 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK 4164 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK 4165 GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4166 Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK- 4167 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK-amide 4168 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK 4169 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK- 4170 amide GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK-amide 4171 Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK 4172 Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK- 4173 amide Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK 4174 GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK-amide 4175 Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK- 4176 amide GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 4177 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4178 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- 4179 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4180 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4181 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4182 amide VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4183 Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4184 VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 4185 Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- 4186 amide GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4187 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4188 GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide 4189 Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK- 4190 amide VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4191 Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4192 VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4193 Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4194 amide NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4195 Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4196 NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4197 Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4198 KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4199 Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4200 KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4201 Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4202 CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4203 Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4204 CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4205 Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4206 GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK 4207 GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK-amide 4208 Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK 4209 Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK- 4210 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 4211 GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 4212 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK 4213 Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK- 4214 amide GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 4215 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK 4216 GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide 4217 Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK- 4218 amide TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4219 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4220 TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4221 Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4222 amide GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4223 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK 4224 GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide 4225 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK- 4226 amide GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4227 GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4228 GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK 4229 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK 4230 GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide 4231 Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK- 4232 amide Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK 4233 GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK-amide 4234 Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK- 4235 amide Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK 4236 GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide 4237 Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK- 4238 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCT 4239 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK 4240 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK 4241 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK 4242 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK 4243 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 4244 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK 4245 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK 4246 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK 4247 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK 4248 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK 4249 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK 4250 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK 4251 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK 4252 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK 4253 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYPK 4254 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYPK 4255 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFPK 4256 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACWPK 4257 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYPK 4258 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK 4259 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK 4260 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK 4261 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK 4262 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK 4263 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK 4264 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK 4265 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC 4266 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC 4267 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC 4268 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC 4269 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC 4270 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC 4271 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHC 4272 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC 4273 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC 4274 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC 4275 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC 4276 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC 4277 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC 4278 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC 4279 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYGG 4280 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG 4281 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG 4282 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG 4283 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG 4284 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 4285 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG 4286 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG 4287 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG 4288 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG 4289 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG 4290 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG 4291 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG 4292 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG 4293 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG 4294 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG 4295 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG 4296 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG 4297 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG 4298 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG 4299 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG 4300 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG 4301 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFGG 4302 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG 4303 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG 4304 GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG 4305 GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG 4306 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG 4307 GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG 4308 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG 4309 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGG 4310 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG 4311 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGG 4312 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK 4313 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK 4314 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK 4315 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 4316 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK 4317 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK 4318 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK 4319 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK 4320 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK 4321 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK 4322 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK 4323 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK 4324 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK 4325 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK 4326 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK 4327 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK 4328 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK 4329 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK 4330 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK 4331 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK 4332 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC 4333 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC 4334 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC 4335 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC 4336 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC 4337 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC 4338 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC 4339 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC 4340 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC 4341 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC 4342 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC 4343 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC 4344 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC 4345 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYGG 4346 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG 4347 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYG 4348 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG 4349 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG 4350 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG 4351 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG 4352 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 4353 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG 4354 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG 4355 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG 4356 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG 4357 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG 4358 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG 4359 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG 4360 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG 4361 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG 4362 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG 4363 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACFGG 4364 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG 4365 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG 4366 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG 4367 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG 4368 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG 4369 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG 4370 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG 4371 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG 4372 GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG 4373 GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG 4374 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP 4375 GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTP 4376 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTP 4377 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK-amide 4378 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK- 4379 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK- 4380 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK- 4381 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK- 4382 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK- 4383 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK- 4384 amide GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK-amide 4385 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK- 4386 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK- 4387 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK- 4388 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK- 4389 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK- 4390 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK- 4391 amide GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK-amide 4392 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK- 4393 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK- 4394 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK- 4395 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACTPK- 4396 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK- 4397 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK- 4398 amide GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC-amide 4399 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC- 4400 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC- 4401 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide 4402 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC- 4403 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide 4404 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC-amide 4405 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC- 4406 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC- 4407 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide 4408 GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC- 4409 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC-amide 4410 GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide 4411 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYGG-amide 4412 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG-amide 4413 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG- 4414 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG- 4415 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG- 4416 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG- 4417 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG- 4418 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCFGG-amide 4419 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYG-amide 4420 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG-amide 4421 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG-amide 4422 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG- 4423 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG- 4424 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG- 4425 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACYGG- 4426 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG- 4427 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG- 4428 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG-amide 4429 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG-amide 4430 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG- 4431 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG- 4432 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG- 4433 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG- 4434 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG- 4435 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGGG-amide 4436 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACFGG-amide 4437 GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG-amide 4438 GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG- 4439 amide GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG- 4440 amide GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG- 4441 amide GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG- 4442 amide GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG- 4443 amide GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG- 4444 amide GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCTPK-amide 4445 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK- 4446 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK- 4447 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK- 4448 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK- 4449 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK- 4450 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK- 4451 amide GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK-amide 4452 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK- 4453 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK- 4454 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK- 4455 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK- 4456 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK- 4457 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK- 4458 amide GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK-amide 4459 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK- 4460 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK- 4461 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK- 4462 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTPK- 4463 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK- 4464 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK- 4465 amide GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC-amide 4466 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC- 4467 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC- 4468 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide 4469 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC- 4470 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide 4471 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC-amide 4472 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC- 4473 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC- 4474 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide 4475 GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC- 4476 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide 4477 GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide 4478 GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCWGG-amide 4479 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG-amide 4480 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG- 4481 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG- 4482 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG- 4483 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 4484 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG- 4485 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG- 4486 amide GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYGG-amide 4487 GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG-amide 4488 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG- 4489 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG- 4490 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG- 4491 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG- 4492 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG- 4493 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG- 4494 amide GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG-amide 4495 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG- 4496 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG- 4497 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG- 4498 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG- 4499 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG- 4500 amide GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG-amide 4501 GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG- 4502 amide GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG- 4503 amide GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG- 4504 amide GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP- 4505 amide GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG- 4506 amide GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACGGG- 4507 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG- 4508 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG- 4509 amide GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACY- 4510 amide Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG- 4511 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG- 4512 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG- 4513 amide GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG 4514 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG-amide 4515 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG 4516 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACY-amide 4517 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG-amide 4518 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG 4519 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYPK 4520 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC 4521 GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG- 4522 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK- 4523 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG- 4524 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG- 4525 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG- 4526 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG- 4527 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG- 4528 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG- 4529 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal] 4530 GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG- 4531 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[4Bip]- 4532 amide GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG- 4533 amide GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCGGG 4534

TABLE 7I Additional useful OSK1 peptide analogs: Combined Ala-11, 12, 27, 29, 30 Substituted Series SEQ ID Sequence/structure NO: GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4535 GVIINVSCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4536 GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK 4537 GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK 4538 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4539 GVIINVSCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4540 GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK 4541 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK 4542 Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK 4543 GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4544 Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK- 4545 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK-amide 4546 Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK 4547 Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK- 4548 amide GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK-amide 4549 Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK 4550 Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK- 4551 amide Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK 4552 GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK-amide 4553 Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK- 4554 amide GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide 4555 Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4556 Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK- 4557 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4558 Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4559 Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4560 amide VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4561 Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4562 VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide 4563 Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK- 4564 amide GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4565 Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4566 GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide 4567 Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK- 4568 amide VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4569 Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4570 VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4571 Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4572 amide NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4573 Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4574 NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4575 Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4576 KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4577 Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4578 KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4579 Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4580 CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4581 Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4582 CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4583 Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4584 GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK 4585 GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK-amide 4586 Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK 4587 Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK- 4588 amide GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK 4589 GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide 4590 Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK 4591 Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK- 4592 amide GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK 4593 Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK 4594 GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK-amide 4595 Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK- 4596 amide TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4597 Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4598 TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4599 Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4600 amide GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4601 Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK 4602 GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide 4603 Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK- 4604 amide GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4605 GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4606 GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK 4607 Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK 4608 GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide 4609 Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK- 4610 amide Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK 4611 GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK-amide 4612 Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK- 4613 amide Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK 4614 GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide 4615 Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK- 4616 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCT 4617 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK 4618 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK 4619 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK 4620 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK 4621 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK 4622 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK 4623 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK 4624 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK 4625 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK 4626 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK 4627 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK 4628 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK 4629 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK 4630 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK 4631 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYPK 4632 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYPK 4633 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFPK 4634 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACWPK 4635 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYPK 4636 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK 4637 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK 4638 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK 4639 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK 4640 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK 4641 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK 4642 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK 4643 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC 4644 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC 4645 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC 4646 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC 4647 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC 4648 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC 4649 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHC 4650 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC 4651 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC 4652 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC 4653 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC 4654 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC 4655 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC 4656 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC 4657 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYGG 4658 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG 4659 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG 4660 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG 4661 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG 4662 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG 4663 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG 4664 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG 4665 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG 4666 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG 4667 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG 4668 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG 4669 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG 4670 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG 4671 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG 4672 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG 4673 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG 4674 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG 4675 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG 4676 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG 4677 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG 4678 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG 4679 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFGG 4680 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG 4681 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG 4682 GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG 4683 GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG 4684 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG 4685 GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG 4686 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG 4687 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGG 4688 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG 4689 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGG 4690 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK 4691 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK 4692 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK 4693 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK 4694 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK 4695 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK 4696 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK 4697 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK 4698 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK 4699 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK 4700 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK 4701 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK 4702 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK 4703 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK 4704 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK 4705 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK 4706 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK 4707 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK 4708 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK 4709 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK 4710 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC 4711 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC 4712 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC 4713 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC 4714 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC 4715 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC 4716 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC 4717 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC 4718 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC 4719 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC 4720 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC 4721 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC 4722 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC 4723 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYGG 4724 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG 4725 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYG 4726 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG 4727 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG 4728 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG 4729 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG 4730 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG 4731 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG 4732 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG 4733 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG 4734 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG 4735 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG 4736 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG 4737 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG 4738 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG 4739 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG 4740 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG 4741 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACFGG 4742 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG 4743 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG 4744 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG 4745 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG 4746 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG 4747 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG 4748 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG 4749 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG 4750 GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG 4751 GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG 4752 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP 4753 GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTP 4754 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTP 4755 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK-amide 4756 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK- 4757 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK- 4758 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK- 4759 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK- 4760 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK- 4761 amide GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK- 4762 amide GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK-amide 4763 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK- 4764 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK- 4765 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK- 4766 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK- 4767 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK- 4768 amide GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK- 4769 amide GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK-amide 4770 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK- 4771 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK- 4772 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK- 4773 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACTPK- 4774 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK- 4775 amide GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK- 4776 amide GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC-amide 4777 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC- 4778 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC- 4779 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC-amide 4780 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC- 4781 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC-amide 4782 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC-amide 4783 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC- 4784 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC- 4785 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC-amide 4786 GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC- 4787 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC-amide 4788 GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC-amide 4789 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYGG-amide 4790 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG-amide 4791 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG- 4792 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG- 4793 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG- 4794 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG- 4795 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG- 4796 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCFGG-amide 4797 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYG-amide 4798 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG-amide 4799 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG-amide 4800 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG- 4801 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG- 4802 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG- 4803 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACYGG- 4804 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG- 4805 amide GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG- 4806 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG-amide 4807 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG-amide 4808 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG- 4809 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG- 4810 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG- 4811 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG- 4812 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG- 4813 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGGG-amide 4814 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACFGG-amide 4815 GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG-amide 4816 GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG- 4817 amide GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG- 4818 amide GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG- 4819 amide GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG- 4820 amide GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG- 4821 amide GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG- 4822 amide GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCTPK-amide 4823 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK- 4824 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK- 4825 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK- 4826 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK- 4827 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK- 4828 amide GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK- 4829 amide GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK-amide 4830 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK- 4831 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK- 4832 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK- 4833 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK- 4834 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK- 4835 amide GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK- 4836 amide GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK-amide 4837 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK- 4838 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK- 4839 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK- 4840 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTPK- 4841 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK- 4842 amide GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK- 4843 amide GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC-amide 4844 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC- 4845 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC- 4846 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC-amide 4847 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC- 4848 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC-amide 4849 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC-amide 4850 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC- 4851 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC- 4852 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC-amide 4853 GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC- 4854 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC-amide 4855 GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC-amide 4856 GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCWGG-amide 4857 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG-amide 4858 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG- 4859 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG- 4860 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG- 4861 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG- 4862 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG- 4863 amide GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG- 4864 amide GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYGG-amide 4865 GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG-amide 4866 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG- 4867 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG- 4868 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG- 4869 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG- 4870 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG- 4871 amide GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG- 4872 amide GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG-amide 4873 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG- 4874 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG- 4875 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG- 4876 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG- 4877 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG- 4878 amide GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG-amide 4879 GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG- 4880 amide GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG- 4881 amide GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG- 4882 amide GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP- 4883 amide GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACGGG- 4884 amide GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACGGG- 4885 amide GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACYGG- 4886 amide GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACGGG- 4887 amide GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACY- 4888 amide Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG- 4889 amide GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG- 4890 amide GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCHCYGG- 4891 amide GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG 4892 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG-amide 4893 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG 4894 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACY-amide 4895 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG-amide 4896 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG 4897 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYPK 4898 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC 4899 GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]GG- 4900 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]PK- 4901 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[2Nal]GG- 4902 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[Cha]GG- 4903 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[MePhe]GG- 4904 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[BiPhA]GG- 4905 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Aib]CYGG- 4906 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Abu]CYGG- 4907 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal] 4908 GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[1Nal]GG- 4909 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[4Bip]- 4910 amide GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[4Bip]GG- 4911 amide GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCGGG 4912 GIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK 4916

TABLE 7J Additional useful OSK1 peptide analogs SEQ Short-hand ID Sequence/Structure designation NO: GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCGC [Gly34]OSK1 4930 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCSC [Ser34]OSK1 4931 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCTC [Thr34]OSK1 4932 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCNC [Asn34]OSK1 4933 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCVC [Val34]OSK1 4934 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCLC [Leu34]OSK1 4935 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCIC [Ile34]OSK1 4936 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCPC [Pro34]OSK1 4937 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCMC [Met34]OSK1 4938 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCQC [Gln34]OSK1 4939 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCKC [Lys34]OSK1 4940 TPK GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCDC [Asp34]OSK1 4941 TPK WVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Trp1]OSK1 4942 WPK GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Trp3]OSK1 4943 TPK GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Trp5]OSK1 4944 TPK [1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMN [1Nal1]OSK1 4945 GKCHCWPK GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCMN [1Nal3]OSK1 4946 GKCHCTPK GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCMN [1Nal5]OSK1 4947 GKCHCTPK GVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCHC [Lys4]OSK1 4948 TPK GVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Lys4, Ala34]OSK1 4949 TPK [1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMN [1Nal1; 4950 GKCACWPK Ala34]OSK1 GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCMN [1Nal3; 4951 GKCACTPK Ala34]OSK1 GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCMN [1Nal5; 4952 GKCACTPK Ala34]OSK1 WVIINVKCKISRQCLEPCKKAGMRFCKCMNGKCAC [Trp1; 4953 WPK Ala34]OSK1 GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Trp3; 4954 TPK Ala34]OSK1 GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Trp5; 4955 TPK Ala34]OSK1 WVWIWVKCKISRQCLEPCKKAGMRFGKCMNGKCAC [Trp1, 3, 5; 4956 TPK Ala34]OSK1 [1Nal]V[1Nal]I[1Nal]VKCKISRQCLEPCKK [1Nal1, 3, 5; 4957 AGMRFGKCMNGKCACTPK Ala34]OSK1 CKISRQCLEPCKKAGMRFGKCMNGKCACTPK Δ1-7, 4958 [Ala34]OSK1 KCKISRQCLEPCKKAGMRFGKCMNGKCACTPK Δ1-6, 4959 [Ala34]OSK1 GVIINVKCKI[1Nal]RQCLEPCKKAGMRFGKCAN [1Nal11; Ala29, 4960 GKCACWPK 34] Osk-1 GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCAC [Arg11; Ala29, 4961 WPK 34] Osk-1 GVIINVKCKISRQCEEPCKKAGMRFGKCANGKCAC [Glu15; Ala29, 4962 WPK 34] Osk-1 GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCAC [Arg11; 4963 WPK Ala34]Osk-1 GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCAC [Glu15; 4964 WPK Ala34]Osk-1 CKIRRQCEEPCKKAGMRFGKCANGKCACTPK Δ1-7, [Arg11; 4965 Glu15; Ala29, 34] OSK1 GVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCAC [Arg11; Glu15; 4966 TPK Ala29, 34]OSK1 CVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCAC [Cys1, 37; Arg11; 4967 TCK Glu15; Ala29, 34]OSK1 GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC [Arg11; Ala12, 4968 TPK 29, 34; Glu15] OSK1 GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC [Arg11; Glu15; 4969 TPK-NH2 Ala12, 29, 34]OSK1- amide Ac- Ac-[Arg11; 4970 GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC Glu15; Ala12, TPK-NH2 29, 34]OSK1- amide GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCAN [1Nal11; 4971 GKCACTPK Glu15; Ala12, 29, 34]OSK1 GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal] [Ala29; 1Nal34] 4972 CWPK Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHC [Ala12; Lys16; 4973 TPK Asp20]Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC [Ala12, 34; Lys16; 4974 TPK Asp20]Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC [Ala12, 29, 34; Lys16; 4975 TPK Asp20]Osk-1 GVIINVKCKIRAQCLKPCKDAGMRFGKCANGKCAC [Arg11; Ala12, 4976 TPK 29, 34; Lys16; Asp20] Osk-1 GVIINVKCKISAQCEKPCKDAGMRFGKCANGKCAC [Ala12, 29, 34; 4977 TPK Glu15, Lys16; Asp20]Osk-1 GVIINVKCKI[1Nal]AQCLKPCKDAGMRFGKCAN [1Nal11; Ala12, 4978 GKCACTPK 29, 34; Lys16; Asp20]Osk-1 GVIINVKCKIRAQCEKPCKDAGMRFGKCANGKCAC [Arg11; Ala12, 4979 TPK 29, 34; Glu15; Lys16; Asp20]Osk-1 GVIINVKCKIRAQCEKPCKDAGMRFGKCMNGKCAC [Arg11; Ala12, 4980 TPK 34; Glu15; Lys16; Asp20]Osk-1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[1Nal] [A12, K16, 4981 CTPK D20, Nal34]- OSK1 GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC [A12, K16, 4982 TPK D20, A34]- OSK1 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[1Nal] [A12, K16, 4983 CTPK D20, A29, Nal34]-OSK1 GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC [A12, K16, 4984 TPK D20, A29, A34]-OSK1 {Acetyl}GVIINVKCKISRQCLEPCK(Glycyl) Ac- 4985 KAGMRFGKCMNGKCACTPK [K(Gly)19, Ala34]- Osk1 {Acetyl}GVIK(Glycyl)NVKCKISRQCLEPCK Ac- 4986 KAGMRFGKCMNGKCACTPK [K(Gly)4, Ala34]- Osk1 {Acetyl}GVIINVKCKISRQCLEPCKKAGMRFGK Ac- 4987 CMNGKCACTPK(Glycyl) [Ala34, K(Gly) 38]-Osk1 GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal] [A29, Nal34]- 4988 CTPK OSK1 CKISRQCLKPCKDAGMRFGKCMNGKCHC{Amide} OSK1[des1- 4989 7, E16K, K20D, des36- 38]-amide GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC OSK1- 4990 TPK K16, D20, A34 GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCAN Osk- 4991 GKC[1Nal]CTPK 1[1Nal11, A12, A29, 1-Nal34] GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCAN [1Nal11, A12, A29, 4992 GKC[1Nal]CTEK 1Nal34, E37] Osk-1 GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCAN Osk-1[1- 4993 GKC[1Nal]CEEK Nal11, A12, E15, A29, 1Nal34, E36, E37] GVIINVKCKI[1Nal]AQCLEPCKKAGFRFGKCAN [1Nal11, A12, F23, 4994 GKC[1Nal]CTPK A29, 1Nal34] Osk-1 GVIINVKCKI[1Nal]AQCLEPCKKAG[Nle]RFG [1Nal11, A12, 4995 KCANGKC[1Nal]CTEK Nle23, A29, 1Nal34, E37]Osk-1 GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCAC [Pro12, Lys16, 4996 TY[Nle] Asp20, Ala34, Tyr37, Nle38] Osk-1- amide GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCAC [P12, Orn16, E20, 4997 TY[Nle] A34, Y37, Nle38] Osk-1- amide NVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal] des1-4, [A29, 4998 CTPK Nal34]-OSK1 NVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK des1-4, [A29, 4999 A34]-OSK1 GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCAC [R11, A29, 5000 TPK A34]-OSK1 GVIINVKCKIRAQCLEPCKKAGMRFGKCANGKCAC [R11, A12, 5001 TPK A29, A34]- OSK1 CKISRQCLEPCKKAGMRFGKCMNGKCACTPK [Ala34]OSK1 5002 (8-35) CKISRQCLEPCKKAGMRFGKCMNGKCAC [Ala34]OSK1 5003 (8-35) CKIRRQCLEPCKKAGMRFGKCANGKCAC [Arg11; Ala29, 5004 34] Osk-1(8-35) CKISAQCLEPCKKAGMRFGKCANGKCAC [Ala12; Ala29, 5005 34] Osk-1(8-35) CKISAQCLEPCKKAGMRFGKCMNGKCAC [Ala12; Ala34] 5006 Osk-1(8-35) GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCM [Dpr^((AOA))4] 5009 NGKCHCTPK Osk1 GVI[Dpr (AOA-PEG) ]NVKCKISRQCLEPCKKAGMRF [Dpr^((AOA)-PEG)) 5010 GKCMNGKCHCTPK 4]Osk1 GCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCK [C2, C37]- 5012 OSK1 SCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCK [S1, C2, C37]- 5013 OSK1 SCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTCK [S1, C2, A34, 5014 C37]-OSK1 SCVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCK Ser-[C1, C37]- 5015 OSK1

Any OSK1 peptide analog that comprises an amino acid sequence selected from SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Tables 7, 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H or 7I, is useful in accordance with the present invention. Any of these can also be derivatized at either its N-terminal or C-terminal, e.g., with a fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon-carbon double bonds, or a derivative thereof such as an ω-amino-fatty acid. (E.g., Mouhat et al., WO 2006/002850 A2, which is incorporated by reference in its entirety). Examples of such fatty acids include valeric acid or (for the C-terminal) ω-amino-valeric acid.

Among useful OSK1 peptide analog sequences of the present invention are analog sequences that introduce amino acid residues that can form an intramolecular covalent bridge (e.g., a disulfide bridge) or non-covalent interactions (e.g. hydrophobic, ionic, stacking) between the first and last beta strand, which may enhance the stability of the structure of the unconjugated or conjugated (e.g., PEGylated) OSK1 peptide analog molecule. Examples of such sequences include SEQ ID NOS: 4985-4987 and 5012-5015.

In some embodiments of the composition of matter, the C-terminal carboxylic acid moiety of the OSK1 peptide analog is replaced with a moiety selected from:

(A) —COOR, where R is independently (C₁-C₈)alkyl, haloalkyl, aryl or heteroaryl;

(B) —C(═O)NRR, where R is independently hydrogen, (C₁-C₈)alkyl, haloalkyl, aryl or heteroaryl; and

(C) —CH₂OR where R is hydrogen, (C₁-C₈) alkyl, aryl or heteroaryl.

“Aryl” is phenyl or phenyl vicinally-fused with a saturated, partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge, the phenyl or bridge being substituted by 0, 1, 2 or 3 substituents selected from C₁₈ alkyl, C₁₄ haloalkyl or halo.

“Heteroaryl” is an unsaturated 5, 6 or 7 membered monocyclic or partially-saturated or unsaturated 6-, 7-, 8-, 9-, 10- or 11 membered bicyclic ring, wherein at least one ring is unsaturated, the monocyclic and the bicyclic rings containing 1, 2, 3 or 4 atoms selected from N, O and S, wherein the ring is substituted by 0, 1, 2 or 3 substituents selected from C₁₈ alkyl, C₁₄ haloalkyl and halo.

In other embodiments of the composition of matter comprising a half-life extending moiety, the OSK1 peptide analog comprises an amino acid sequence of the formula:

G¹V²I³I⁴N⁵V⁶K⁷C⁸K⁹I¹⁰X_(aa) ¹¹X_(aa) ¹² SEQ ID NO: 5011 Q¹³C¹⁴X_(aa) ¹⁵X_(aa) ¹⁶P¹⁷C¹⁸X_(aa) ¹⁹X_(aa) ²⁰ A²¹G²²M²³R²⁴F²⁵G²⁶X_(aa) ²⁷C²⁸X_(aa) ²⁹ X_(aa) ³⁰G³¹X_(aa) ³²C³³X_(aa) ³⁴C³⁵X_(aa) ³⁶ X_(aa) ³⁷X_(aa) ³⁸

wherein:

amino acid residues 1 through 7 are optional (Thus, the OSK1 peptide analog optionally includes residues 1-7 as indicated above in SEQ ID NO:5011, or a N-terminal truncation leaving present residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a N-terminal truncation wherein all of residues 1-7 are entirely absent.);

-   -   X_(aa) ¹¹ is a neutral, basic, or acidic amino acid residue         (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,         Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and         4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,         Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,         Asn, Gln, Glu, Asp, α-aminoadipic acid, and         para-carboxyl-phenylalanine);     -   X_(aa) ¹² is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ¹⁵ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ¹⁶ is a neutral or basic amino acid residue (e.g., Lys,         His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,         2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,         Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,         Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);     -   X_(aa) ¹⁹ is a neutral or basic amino acid residue (e.g., Lys,         His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,         2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,         Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,         Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);     -   X_(aa) ²⁰ is a neutral or basic amino acid residue (e.g., Lys,         His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,         2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitruiline, His,         Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,         Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);     -   X_(aa) ²⁷ is a neutral, basic, or acidic amino acid residue         (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,         Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and         4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,         Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,         Asn, Gln, Glu, Asp, α-aminoadipic acid, and         para-carboxyl-phenylalanine);     -   X_(aa) ²⁹ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ³⁰ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ³² is a neutral, basic, or acidic amino acid residue         (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,         Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and         4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,         Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,         Asn, Gln, Glu, Asp, α-aminoadipic acid, and         para-carboxyl-phenylalanine);     -   X_(aa) ³⁴ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ³⁶ is optional, and if present, is a neutral amino acid         residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,         D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,         Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;     -   X_(aa) ³⁷ is optional, and if present, is a neutral amino acid         residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,         D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,         Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and     -   X_(aa) ³⁸ is optional, and if present, is a basic amino acid         residue (e.g., Lys, His, Orn, D-Orn, Arg, N^(α) Methyl-Arg;         homoarginine, Cit, N^(α)-Methyl-Cit, Homocitrulline, Guf, and         4-Amino-Phe).

In some other embodiments of the composition of matter comprising a half-life extending moiety, the OSK1 peptide analog comprises an amino acid sequence of the formula:

G¹V²I³I⁴N⁵V⁶K⁷C⁸K⁹I¹⁰X_(aa) ¹¹X_(aa) ¹² SEQ ID NO: 4913 Q¹³C¹⁴L¹⁵X_(aa) ¹⁶P¹⁷C¹⁸K¹⁹X_(aa) ²⁰A²¹ G²²M²³R²⁴F²⁵G²⁶X_(aa) ²⁷C²⁸X_(aa) ²⁹X_(aa) ³⁰ G³¹K³²C³³X_(aa) ³⁴C³⁵X_(aa) ³⁶X_(aa) ³⁷X_(aa) ³⁸

wherein:

-   -   amino acid residues 1 to 7 are optional (Thus, the OSK1 peptide         analog optionally includes residues 1-7 as indicated above in         SEQ ID NO:4913, or a N-terminal truncation leaving present         residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a         N-terminal truncation wherein all of residues 1-7 are entirely         absent.);     -   X_(aa) ¹¹ is a neutral, basic or acidic amino acid residue         (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,         Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and         4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,         Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,         Asn, Gln, Glu, Asp, α-aminoadipic acid, and         para-carboxyl-phenylalanine);     -   X_(aa) ¹² is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ¹⁶ is a neutral or basic amino acid residue (e.g., Lys,         His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,         2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,         Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,         Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);     -   X_(aa) ²⁰ is a neutral or basic amino acid residue (e.g., Lys,         His, Arg, Trp, Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal,         2-Nal, Orn, D-Orn, Cit, N^(α)-Methyl-Cit, Homocitrulline, His,         Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro,         Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);     -   X_(aa) ²⁷ is a neutral, basic, or acidic amino acid residue         (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,         Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and         4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,         Arg, N^(α) Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,         Asn, Gln, Glu, Asp, α-aminoadipic acid, and         para-carboxyl-phenylalanine);     -   X_(aa) ²⁹ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ³⁰ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ³⁵ is a neutral or acidic amino acid residue (e.g., Ala,         Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz,         Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp,         α-aminoadipic acid, and para-carboxyl-phenylalanine);     -   X_(aa) ³⁶ is optional, and if present, is a neutral amino acid         residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,         D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,         Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;     -   X_(aa) ³⁷ is optional, and if present, is a neutral amino acid         residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic,         D-Tic, D-Pro, Thz, N^(α)-Methyl-Cit, Homocitrulline, Aib, Sar,         Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and     -   X_(aa) ³⁸ is optional, and if present, is a basic amino acid         residue (e.g., Lys, His, Orn, D-Orn, Arg, N^(α) Methyl-Arg;         homoarginine, Cit, N^(α)-Methyl-Cit, Homocitruiline, Guf, and         4-Amino-Phe).

TABLE 8 Pi2 peptide and PiP2 s peptide analog equences Short- hand desig- SEQ ID Sequence/structure nation NO: TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2 17 TISCTNPXQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2-X8 299 TISCTNPAQCYPHCKKETGYPNAKCMNRKCKCFGR Pi2-A8 300 TISCTNPKQCYPHCXKETGYPNAKCMNRKCKCFGR Pi2-X15 301 TISCTNPKQCYPHCAKETGYPNAKCMNRKCKCFGR Pi2-A15 302 TISCTNPKQCYPHCKXETGYPNAKCMNRKCKCFGR Pi2-X16 303 TISCTNPKQCYPHCKAETGYPNAKCMNRKCKCFGR Pi2-A16 304 TISCTNPKQCYPHCKKETGYPNAXCMNRKCKCFGR Pi2-X24 305 TISCTNPKQCYPHCKKETGYPNAACMNRKCKCFGR Pi2-A24 306 TISCTNPKQCYPHCKKETGYPNAKCMNXKCKCFGR Pi2-X28 307 TISCTNPKQCYPHCKKETGYPNAKCMNAKCKCFGR Pi2-A28 308 TISCTNPKQCYPHCKKETGYPNAKCMNRXCKCFGR Pi2-X29 309 TISCTNPKQCYPHCKKETGYPNAKCMNRACKCFGR Pi2-A29 310 TISCTNPKQCYPHCKKETGYPNAKCMNRKCXCFGR Pi2-X31 311 TISCTNPKQCYPHCKKETGYPNAKCMNRKCACFGR Pi2-A31 312 TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGX Pi2-X35 313 TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGA Pi2-A35 314 TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFG Pi2-d35 315

TABLE 9 Anuroctoxin (AnTx) peptide and peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: ZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCF Anuroctoxin 62 NCK (AnTx) KECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1 316 XECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1, X2 317 AECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK AnTx-d1, A2 318

TABLE 10 Noxiustoxin (NTX) peptide and NTX peptide analog sequences Short- hand SEQ desig- ID Sequence/structure nation NO: TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX 30 TIINVACTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX-A6 319 TIINVKCTSPKQCSKPCKELYGSSRGAKCMNGKCKCYNN NTX- 320 R25 TIINVKCTSSKQCSKPCKELYGSSAGAKCMNGKCKCYNN NTX- 321 S10 TIINVKCTSPKQCWKPCKELYGSSAGAKCMNGKCKCYNN NTX- 322 W14 TIINVKCTSPKQCSKPCKELYGSSGAKCMNGKCKCYNN NTX- 323 A25d TIINVKCTSPKQCSKPCKELFGVDRGKCMNGKCKCYNN NTX- 324 IbTx1 TIINVKCTSPKQCWKPCKELFGVDRGKCMNGKCKCYN NTX- 325 IBTX2

TABLE 11 Kaliotoxin1 (KTX1) peptide and KTX1 peptide analog sequences Short- hand SEQ desig- ID Sequence/structure nation NO: GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK KTX1 24 VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK KTX2 326 GVEINVSCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK KTX1- 327 S7 GVEINVACSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK KTX1- 328 A7

TABLE 12 IKCa1 inhibitor peptide sequences Short- hand SEQ desig- ID Sequence/structure nation NO: VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX 20 QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx 36 QFTQESCTASNQCWSICKRLHNTNRGKCMNKKCRCYS ChTx-Lq2 329

TABLE 13 Maurotoxin (MTx) peptide amd MTx peptide analog sequences Short- hand SEQ desig- ID Sequence/structure nation NO: VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX 20 VSCAGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A4 330 VSCTGAKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A6 331 VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A7 332 VSCTGSKDCAAPCRKQTGCPNAKCINKSCKCYGC MTX-A10 333 VSCTGSKDCYAPCQKQTGCPNAKCINKSCKCYGC MTX-Q14 334 VSCTGSKDCYAPCRQQTGCPNAKCINKSCKCYGC MTX-Q15 335 VSCTGSKDCYAPCQQQTGCPNAKCINKSCKCYGC MTX- 336 Q14, 15 VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYAC MTX-A33 337 VSCTGSKDCYAPCRKQTGCPYGKCMNRKCKCNRC MTX- 338 HsTx1 VSCTGSKDCYAACRKQTGCANAKCINKSCKCYGC MTX- 339 A12, 20 VSCTGSKDCYAPCRKQTGX^(M19) MTX- 340 PNAKCINKSCKCYGX^(M34) X19, 34 VSCTGSKDCYAPCRKQTGSPNAKCINKSCKCYGS MTX- 341 S19, 34 VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A7 342 VVIGQRCTGSKDCYAPCRKQTGCPNAKCINK TsK-MTX 343 SCKCYGC VSCRGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-R4 1301 VSCGGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-G4 1302 VSCTTSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-T5 1304 VSCTASKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A5 1305 VSCTGTKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-T6 1306 VSCTGPKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-P6 1307 VSCTGSKDCGAPCRKQTGCPNAKCINKSCKCYGC MTX-G10 1309 VSCTGSKDCYRPCRKQTGCPNAKCINKSCKCYGC MTX-R11 1311 VSCTGSKDCYDPCRKQTGCPNAKCINKSCKCYGC MTX-D11 1312 VSCTGSKDCYAPCRKRTGCPNAKCINKSCKCYGC MTX-R16 1315 VSCTGSKDCYAPCRKETGCPNAKCINKSCKCYGC MTX-E16 1316 VSCTGSKDCYAPCRKQTGCPYAKCINKSCKCYGC MTX-Y21 1317 VSCTGSKDCYAPCRKQTGCPNSKCINKSCKCYGC MTX-S22 1318 VSCTGSKDCYAPCRKQTGCPNGKCINKSCKCYGC MTX-G22 1319 VSCTGSKDCYAPCRKQTGCPNAKCINRSCKCYGC MTX-R27 1320 VSCTGSKDCYAPCRKQTGCPNAKCINKTCKCYGC MTX-T28 1321 VSCTGSKDCYAPCRKQTGCPNAKCINKMCKCYGC MTX-M28 1322 VSCTGSKDCYAPCRKQTGCPNAKCINKKCKCYGC MTX-K28 1323 VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCNGC MTX-N32 1324 VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYRC MTX-R33 1325 VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCS MTX-S35 1326 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-d1 1327 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCS MTX- 1328 S35 d1 VSCTGSKDCYAPCAKQTGCPNAKCINKSCKCYGC MTX-A14 1329 VSCTGSKDCYAPCRAQTGCPNAKCINKSCKCYGC MTX-A15 1330 VSCTGSKDCYAPCRKQTGCPNAACINKSCKCYGC MTX-A23 1331 VSCTGSKDCYAPCRKQTGCPNAKCINASCKCYGC MTX-A27 1332 VSCTGSKDCYAPCRKQTGCPNAKCINKSCACYGC MTX-A30 1333 VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCAGC MTX-A32 1334 ASCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-A1 1335 MSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC MTX-M1 1336

In Table 13 and throughout this specification, X^(m19) and X^(m34) are each independently nonfunctional residues.

TABLE 14 Charybdotoxin(ChTx) peptide and ChTx peptide analog sequences Short-hand Sequence/structure designation SEQ ID NO: QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx 36 QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKECRCYS ChTx-E32 59 QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKDCRCYS ChTx-D32 344 CTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-d1-d6 345 QFTNVSCTTSKECWSVCQRLFGVDRGKCMGKKCRCYQ ChTx-IbTx 346 QFTNVSCTTSKECWSVCQRLHNTSRGKCMNGKCRCYS ChTx-G31 1369 QFTNVSCTTSKECLSVCQRLHNTSRGKCMNKKCRCYS ChTx-L14 1370 QFTNVSCTTSKECASVCQRLHNTSRGKCMNKKCRCYS ChTx-A14 1371 QFTNVSCTTSKECWAVCQRLHNTSRGKCMNKKCRCYS ChTx-A15 1372 QFTNVSCTTSKECWPVCQRLHNTSRGKCMNKKCRCYS ChTx-P15 1373 QFTNVSCTTSKECWSACQRLHNTSRGKCMNKKCRCYS ChTx-A16 1374 QFTNVSCTTSKECWSPCQRLHNTSRGKCMNKKCRCYS ChTx-P16 1375 QFTNVSCTTSKECWSVCQRLHNTSAGKCMNKKCRCYS ChTx-A25 1376 QFTNVACTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A6 1377 QFTNVKCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-K6 1378 QFTNVSCTTAKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A10 1379 QFTNVSCTTPKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-P10 1380 QFTNVSCTTSKACWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A12 1381 QFTNVSCTTSKQCWSVCQRLHNTSRGKCMNKKCRCYS ChTx-Q12 1382 AFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A1 1383 TFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-T1 1384 QATNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A2 1385 QITNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-I2 1386 QFANVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-A3 1387 QFINVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS ChTx-I3 1388 TIINVKCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP ChTx-MgTx 1389 TIINVSCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP ChTx-MgTx-b 1390

TABLE 15 SKCa inhibitor peptide sequences SEQ Short-hand ID Sequence/structure designation NO: CNCKAPETALCARRCQQHG Apamin 68 AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH ScyTx 51 AVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG BmP05 50 TVCNLRRCQLSCRSLGLLGKCIGVKCECVKH P05 52 AFCNLRRCELSCRSLGLLGKCIGEECKCVPY Tamapin 53 VSCEDCPEHCSTQKAQAKCDNDKCVCEPI P01 16 VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDC TsK 47

TABLE 16 Apamin peptide and peptide analog inhibitor sequences Short-hand Sequence/structure designation SEQ ID NO: CNCKAPETALCARRCQQHG Apamin (Ap) 68 CNCXAPETALCARRCQQHG Ap-X4 348 CNCAAPETALCARRCQQHG Ap-A4 349 CNCKAPETALCAXRCQQHG Ap-X13 350 CNCKAPETALCAARCQQHG Ap-A13 351 CNCKAPETALCARXCQQHG Ap-X14 352 CNCKAPETALCARACQQHG Ap-A14 353

TABLE 17 Scyllatoxin (ScyTx), BmP05, P05, Tamapin, P01 peptide and peptide analog inhibitor sequences Short-hand SEQ Sequence/structure designation ID NO: AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH ScyTx 51 AFCNLRRCQLSCRSLGLLGKCIGDKCECVKH ScyTx-R7 354 AFCNLRMCQLSCRSLGLLGKCMGKKCRCVKH ScyTx-IbTx 355 AFSNLRMCQLSCRSLGLLGKSIGDKCECVKH ScyTx-C/S 356 AFCNLRRCELSCRSLGLLGKCIGEECKCVPY Tamapin 53

TABLE 18 BKCa inhibitor peptide sequences Short-hand Sequence/structure designation SEQ ID NO: QFTDVDCSVSKECWSVCKD IbTx 38 LFGVDRGKCMGKKCRCYQ TFIDVDCTVSKECWAPCKAA Slotoxin 39 FGVDRGKCMGKKCKCYV (SloTx) QFTDVKCTGSKQCWPVCK BmTx1 40 QMFGKPNGKCMNGKCRCYS WCSTCLDLACGASRECYDPCFKAF BuTx 41 GRAHGKCMNKCRCYTN FGLIDVKCFASSECWTACKKV MartenTx 35 TGSGQGKCQNNQCRCY ITINVKCTSPQQCLRPCKDRFGQ CIITx1 29 HAGGKCINGKCKCYP

TABLE 19 IbTx, Slotoxin, BmTx1, & BuTX (Slotoxin family) peptide and peptide analog inhibitor sequences Short-hand Sequence/structure designation SEQ ID NO: QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx 38 QFTDVDCSVSXECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx-X11 357 QFTDVDCSVSAECWSVCKDLFGVDRGKCMGKKCRCYQ IbTx-A11 358 QFTDVDCSVSKECWSVCXDLFGVDRGKCMGKKCRCYQ IbTx-X18 359 QFTDVDCSVSKECWSVCADLFGVDRGKCMGKKCRCYQ IbTx-A18 360 QFTDVDCSVSKECWSVCKDLFGVDXGKCMGKKCRCYQ IbTx-X25 361 QFTDVDCSVSKECWSVCKDLFGVDAGKCMGKKCRCYQ IbTx-A25 362 QFTDVDCSVSKECWSVCKDLFGVDRGXCMGKKCRCYQ IbTx-X27 363 QFTDVDCSVSKECWSVCKDLFGVDRGACMGKKCRCYQ IbTx-A27 364 QFTDVDCSVSKECWSVCKDLFGVDRGKCMGXKCRCYQ IbTx-X31 365 QFTDVDCSVSKECWSVCKDLFGVDRGKCMGAKCRCYQ IbTx-A31 366 QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKXCRCYQ IbTx-X32 367 QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKACRCYQ IbTx-A32 368 QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCXCYQ IbTx-X34 369 QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCACYQ IbTx-A34 370 QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1 371 QFTDVXCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-X6 372 QFTDVACTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-A6 373 QFTDVKCTGSXQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-X11 374 QFTDVKCTGSAQCWPVCKQMFGKPNGKCMNGKCRCYS BmTx1-A11 375 QFTDVKCTGSKQCWPVCXQMFGKPNGKCMNGKCRCYS BmTx1-X18 376 QFTDVKCTGSKQCWPVCAQMFGKPNGKCMNGKCRCYS BmTx1-A18 377 QFTDVKCTGSKQCWPVCKQMFGXPNGKCMNGKCRCYS BmTx1-X23 378 QFTDVKCTGSKQCWPVCKQMFGAPNGKCMNGKCRCYS BmTx1-A23 379 QFTDVKCTGSKQCWPVCKQMFGKPNGXCMNGKCRCYS BmTx1-X27 380 QFTDVKCTGSKQCWPVCKQMFGKPNGACMNGKCRCYS BmTx1-A27 381 QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGXCRCYS BmTx1-X32 382 QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGARCYS BmTx1-A32 383 QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCXYS BmTx1-X34 384 QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCAYS BmTx1-A34 385 WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN BuTx 386 WCSTCLDLACGASXCYDPCFKAFGRAHGKCMNNKCRCYTN BuTx-X14 387 WCSTCLDLACGASACYDPCFKAFGRAHGKCMNNKCRCYTN BuTx-A14 388 WCSTCLDLACGASRECYDPCFXFGRAHGKCMNNKCRCYTN BuTx-X22 389 WCSTCLDLACGASRECYDPCFAGRAHGKCMNNKCRCYTN BuTx-A22 390 WCSTCLDLACGASRECYDPCFKAFGXHGKCMNNKCRCYTN BuTx-X26 391 WCSTCLDLACGASRECYDPCFKAFGAHGKCMNNKCRCYTN BuTx-A26 392 WCSTCLDLACGASRECYDPCFKAFGRAHGXMNNKCRCYTN BuTx-X30 393 WCSTCLDLACGASRECYDPCFKAFGRAHGAMNNKCRCYTN BuTx-A30 394 WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNXRCYTN BuTx-X35 395 WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNARCYTN BuTx-A35 396 WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCXYTN BuTx-X37 397 WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCAYTN BuTx-A37 398

TABLE 20 Martentoxin peptide and peptide analog inhibitor sequences Short-hand Sequence/structure designation SEQ ID NO: FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx 35 FGLIDVXCFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx-X7 399 FGLIDVACFASSECWTACKKVTGSGQGKCQNNQCRCY MartenTx-A7 400 FGLIDVKCFASSECWTACXKVTGSGQGKCQNNQCRCY MartenTx-X19 401 FGLIDVKCFASSECWTACAKVTGSGQGKCQNNQCRCY MartenTx-A19 402 FGLIDVKCFASSECWTACKXVTGSGQGKCQNNQCRCY MartenTx-X20 403 FGLIDVKCFASSECWTACKAVTGSGQGKCQNNQCRCY MartenTx-A20 404 FGLIDVKCFASSECWTACKKVTGSGQGXCQNNQCRCY MartenTx-X28 405 FGLIDVKCFASSECWTACKKVTGSGQGACQNNQCRCY MartenTx-A28 406 FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCXCY MartenTx-X35 407 FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCACY MartenTx-A35 408

TABLE 21 N type Ca²⁺ channel inhibitor peptide sequences SEQ Short-hand ID Sequence/structure designation NO: CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA 65 CKSPGSSCSPTSYNCCRSCNPYTKRCY GVIA 64 CKSKGAKCSKLMYDCCTGSCSGTVGRC CVIA 409 CKLKGQSCRKTSYDCCSGSCGRSGKC SVIB 347 AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1 66 CKGKGASCRKTMYDCCRGSCRSGRC CVIB 1364 CKGKGQSCSKLMYDCCTGSCSRRGKC CVIC 1365 CKSKGAKCSKLMYDCCSGSCSGTVGRC CVID 1366 CLSXGSSCSXTSYNCCRSCNXYSRKCY TVIA 1367

TABLE 22 ωMVIIA peptide and peptide analog inhibitor sequences Short-hand Sequence/structure designation SEQ ID NO: CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA 65 CXGKGAKCSRLMYDCCTGSCRSGKC MVIIA-X2 410 CAGKGAKCSRLMYDCCTGSCRSGKC MVIIA-A2 411 CKGXGAKCSRLMYDCCTGSCRSGKC MVIIA-X4 412 CKGAGAKCSRLMYDCCTGSCRSGKC MVIIA-A4 413 CKGKGAXCSRLMYDCCTGSCRSGKC MVIIA-X7 414 CKGKGAACSRLMYDCCTGSCRSGKC MVIIA-A7 415 CKGKGAKCSXLMYDCCTGSCRSGKC MVIIA-X10 416 CKGKGAKCSALMYDCCTGSCRSGKC MVIIA-A10 417 CKGKGAKCSRLMYDCCTGSCXSGKC MVIIA-X21 418 CKGKGAKCSRLMYDCCTGSCASGKC MVIIA-A21 419 CKGKGAKCSRLMYDCCTGSCRSGXC MVIIA-X24 420 CKGKGAKCSRLMYDCCTGSCRSGAC MVIIA-A24 421

TABLE 23 □GVIA peptide and peptide analog inhibitor sequences Short-hand Sequence/structure designation SEQ ID NO: CKSPGSSCSPTSYNCCRSCNPYTKRCY GVIA 64 CXSPGSSCSPTSYNCCRSCNPYTKRCY GVIA-X2 422 CASPGSSCSPTSYNCCRSCNPYTKRCY GVIA-A2 423 CKSPGSSCSPTSYNCCXSCNPYTKRCY GVIA-X17 424 CKSPGSSCSPTSYNCCASCNPYTKRCY GVIA-A17 425 CKSPGSSCSPTSYNCCRSCNPYTXRCY GVIA-X24 426 CKSPGSSCSPTSYNCCRSCNPYTARCY GVIA-A24 427 CKSPGSSCSPTSYNCCRSCNPYTKXCY GVIA-X25 428 CKSPGSSCSPTSYNCCRSCNPYTKACY GVIA-A25 429

TABLE 24 Ptu1 peptide and peptide analog inhibitor sequences SEQ Short-hand ID Sequence/structure designation NO: AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1 66 AEXDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1-X3 430 AEADCIAPGAPCFGTDKPCCNPRAWCSSYANKCL Ptu1-A3 431 AEKDCIAPGAPCFGTDXPCCNPRAWCSSYANKCL Ptu1-X17 432 AEKDCIAPGAPCFGTDAPCCNPRAWCSSYANKCL Ptu1-A17 433 AEKDCIAPGAPCFGTDKPCCNPXAWCSSYANKCL Ptu1-X23 434 AEKDCIAPGAPCFGTDKPCCNPAAWCSSYANKCL Ptu1-A23 435 AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANXCL Ptu1-X32 436 AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANACL Ptu1-A32 437

TABLE 25 Thrixopelma pruriens (ProTx1) and ProTx1 peptide analogs and other T type Ca²⁺ channel inhibitor peptide sequences Short-hand SEQ Sequence/structure designation ID NO: ECRYWLGGCSAGQTCCKHLVCSRRHGWC ProTx1 56 VWDGTFS ECXYWLGGCSAGQTCCKHLVCSRRHGWC ProTx1-X3 438 VWDGTFS ECAYWLGGCSAGQTCCKHLVCSRRHGWC ProTx1-A3 439 VWDGTFS ECRYWLGGCSAGQTCCXHLVCSRRHGWC ProTx1-X17 440 VWDGTFS ECRYWLGGCSAGQTCCAHLVCSRRHGWC ProTx1-A17 441 VWDGTFS ECRYWLGGCSAGQTCCKHLVCSXRHGWC ProTx1-X23 442 VWDGTFS ECRYWLGGCSAGQTCCKHLVCSARHGWC ProTx1-A23 443 VWDGTFS ECRYWLGGCSAGQTCCKHLVCSRXHGWC ProTx1-X24 444 VWDGTFS ECRYWLGGCSAGQTCCKHLVCSRAHGWC ProTx1-A24 445 VWDGTFS KIDGYPVDYW NCKRICWYNN KYCNDLCKGL Kurtoxin 1276 KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA

TABLE 26 BeKM1 M current inhibitor peptide and BeKM1 peptide analog sequences SEQ Short-hand ID Sequence/structure designation NO: RPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1 63 PTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-d1 446 XPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-X1 447 APTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-A1 448 RPTDIXCSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-X6 449 RPTDIACSESYQCFPVCKSRFGKTNGRCVNGFCDCF BeKM1-A6 450 RPTDIKCSESYQCFPVCXSRFGKTNGRCVNGFCDCF BeKM1-X18 451 RPTDIKCSESYQCFPVCASRFGKTNGRCVNGFCDCF BeKM1-A18 452 RPTDIKCSESYQCFPVCKSXFGKTNGRCVNGFCDCF BeKM1-X20 453 RPTDIKCSESYQCFPVCKSAFGKTNGRCVNGFCDCF BeKM1-A20 454 RPTDIKCSESYQCFPVCKSRFGXTNGRCVNGFCDCF BeKM1-X23 455 RPTDIKCSESYQCFPVCKSRFGATNGRCVNGFCDCF BeKM1-A23 456 RPTDIKCSESYQCFPVCKSRFGKTNGXCVNGFCDCF BeKM1-X27 457 RPTDIKCSESYQCFPVCKSRFGKTNGACVNGFCDCF BeKM1-A27 458

TABLE 27 Na⁺ channel inhibitor peptide sequences Short-hand SEQ ID Sequence/structure designation NO: QRCCNGRRGCSSRWCRDHSRCC SmIIIa 459 RDCCTOOKKCKDRQCKOQRCCA μ-GIIIA 460 RDCCTOORKCKDRRCKOMRCCA μ-GIIIB 461 ZRLCCGFOKSCRSRQCKOHRCC μ-PIIIA 462 ZRCCNGRRGCSSRWCRDHSRCC μ-SmIIIA 463 ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV μO-MrVIA 464 ACSKKWEYCIVPIIGFIYCCPGLICGPFVCV μO-MrVIB 465 EACYAOGTFCGIKOGLCCSEFCLPGVCFG δ-PVIA 466 DGCSSGGTFCGIHOGLCCSEFCFLWCITFID δ-SVIE 467 WCKQSGEMCNLLDQNCCDGYCIVLVCT δ-TxVIA 468 VKPCRKEGQLCDPIFQNCCRGWNCVLFCV δ-GmVIA 469

TABLE 28 Cl⁻ channel inhibitor peptide sequences Short-hand Sequence/structure designation SEQ ID NO: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR CTX 67 MCMPCFTTDHQMAXKCDDCCGGKGRGKCYGPQCLCR CTX-X14 470 MCMPCFTTDHQMAAKCDDCCGGKGRGKCYGPQCLCR CTX-A14 471 MCMPCFTTDHQMARXCDDCCGGKGRGKCYGPQCLCR CTX-X15 472 MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR CTX-A15 473 MCMPCFTTDHQMARKCDDCCGGXGRGKCYGPQCLCR CTX-X23 474 MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCR CTX-A23 475 MCMPCFTTDHQMARKCDDCCGGKGXGKCYGPQCLCR CTX-X25 476 MCMPCFTTDHQMARKCDDCCGGKGAGKCYGPQCLCR CTX-A25 477 MCMPCFTTDHQMARKCDDCCGGKGRGXCYGPQCLCR CTX-X27 478 MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCR CTX-A27 479 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCX CTX-X36 480 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCA CTX-A36 481 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLC CTX-d36 482 QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNRE Bm-12b 483 QTDGCGPCFTTDANMAXKCRECCGGNGKCFGPQCLCNRE Bm-12b-X17 484 QTDGCGPCFTTDANMAAKCRECCGGNGKCFGPQCLCNRE Bm-12b-A17 485 QTDGCGPCFTTDANMARXCRECCGGNGKCFGPQCLCNRE Bm-12b-X18 486 QTDGCGPCFTTDANMARACRECCGGNGKCFGPQCLCNRE Bm-12b-A18 487 QTDGCGPCFTTDANMARKCXECCGGNGKCFGPQCLCNRE Bm-12b-X20 488 QTDGCGPCFTTDANMARKCAECCGGNGKCFGPQCLCNRE Bm-12b-A20 489 QTDGCGPCFTTDANMARKCRECCGGNGXCFGPQCLCNRE Bm-12b-X28 490 QTDGCGPCFTTDANMARKCRECCGGNGACFGPQCLCNRE Bm-12b-A28 491 QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNXE Bm-12b-X38 492 QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNAE Bm-12b-A38 493

TABLE 29 Kv2.1 inhibitor peptide sequences SEQ Short-hand ID Sequence/structure designation NO: ECRYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1 494 ECXYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-X3 495 ECAYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-A3 496 ECRYLFGGCXTTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-X10 497 ECRYLFGGCATTSDCCKHLGCKFRDKYCAWDFTFS HaTx1-A10 498 ECRYLFGGCKTTSDCCXHLGCKFRDKYCAWDFTFS HaTx1-X17 499 ECRYLFGGCKTTSDCCAHLGCKFRDKYCAWDFTFS HaTx1-A17 500 ECRYLFGGCKTTSDCCKHLGCXFRDKYCAWDFTFS HaTx1-X22 501 ECRYLFGGCKTTSDCCKHLGCAFRDKYCAWDFTFS HaTx1-A22 502 ECRYLFGGCKTTSDCCKHLGCKFXDKYCAWDFTFS HaTx1-X24 503 ECRYLFGGCKTTSDCCKHLGCKFADKYCAWDFTFS HaTx1-A24 504 ECRYLFGGCKTTSDCCKHLGCKFRDXYCAWDFTFS HaTx1-X26 505 ECRYLFGGCKTTSDCCKHLGCKFRDAYCAWDFTFS HaTx1-A26 506

TABLE 30 Kv4.3 & Kv4.2 inhibitor peptide sequences Short-hand SEQ Sequence/structure designation ID NO: YCQKWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2 57 YCQXWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2-X4 507 YCQAWMWTCDEERKCCEGLVCRLWCKRIINM PaTx2-A4 508 YCQKWMWTCDEEXKCCEGLVCRLWCKRIINM PaTx2-X13 509 YCQKWMWTCDEEAKCCEGLVCRLWCKRIINM PaTx2-A13 510 YCQKWMWTCDEERXCCEGLVCRLWCKRIINM PaTx2-X14 511 YCQKWMWTCDEERACCEGLVCRLWCKRIINM PaTx2-A14 512 YCQKWMWTCDEERKCCEGLVCXLWCKRIINM PaTx2-X22 513 YCQKWMWTCDEERKCCEGLVCALWCKRIINM PaTx2-A22 514 YCQKWMWTCDEERKCCEGLVCRLWCXRIINM PaTx2-X26 515 YCQKWMWTCDEERKCCEGLVCRLWCARIINM PaTx2-A26 516 YCQKWMWTCDEERKCCEGLVCRLWCKXIINM PaTx2-X27 517 YCQKWMWTCDEERKCCEGLVCRLWCKAIINM PaTx2-A27 518

TABLE 31 nACHR channel inhibitor peptide sequences SEQ Short-hand ID Sequence/structure designation NO: GCCSLPPCAANNPDYC PnIA 519 GCCSLPPCALNNPDYC PnIA-L10 520 GCCSLPPCAASNPDYC PnIA-S11 521 GCCSLPPCALSNPDYC PnIB 522 GCCSLPPCAASNPDYC PnIB-A10 523 GCCSLPPCALNNPDYC PnIB-N11 524 GCCSNPVCHLEHSNLC MII 525 GRCCHPACGKNYSC α-MI 526 RD(hydroxypro)CCYHPTCNMSNPQIC α-EI 527 GCCSYPPCFATNPDC α-AuIB 528 RDPCCSNPVCTVHNPQIC α-PIA 529 GCCSDPRCAWRC α-ImI 530 ACCSDRRCRWRC α-ImII 531 ECCNPACGRHYSC α-GI 532 GCCGSY(hydroxypro)NAACH(hydroxypro) αA-PIVA 533 CSCKDR (hydroxypro)SYCGQ GCCPY(hydroxypro)NAACH(hydroxypro) αA-EIVA 534 CGCKVGR(hydroxypro)(hydroxypro)YCDR (hydroxypro)S GG H(hydroxypro)(hydroxypro)CCLYGKCRRY ψ-PIIIE 535 (hydroxypro)GCSSASCCQR GCCSDPRCNMNNPDYC EpI 536 GCCSHPACAGNNQHIC GIC 537 IRD(γ-carboxyglu) CCSNPACRVNN GID 538 (hydroxypro)HVC GGCCSHPACAANNQDYC AnIB 539 GCCSYPPCFATNSDYC AuIA 540 GCCSYPPCFATNSGYC AuIC 541

TABLE 32 Agelenopsis aperta (Agatoxin) toxin peptides and peptide analogs and other Ca²⁺ channel inhibiter peptides SEQ Short-hand ID Sequence/structure designation NO: KKKCIAKDYG RCKWGGTPCC RGRGCICSIM ω-Aga-IVA 959 GTNCECKPRL IMEGLGLA EDNCIAEDYG KCTWGGTKCC RGRPCRCSMI ω-Aga-IVB 960 GTNCECTPRL IMEGLSFA SCIDIGGDCD GEKDDCQCCR RNGYCSCYSL ω-Aga-IIIA 961 FGYLKSGCKC VVGTSAEFQG ICRRKARQCY NSDPDKCESH NKPKRR SCIDIGGDCD GEKDDCQCCR RNGYCSCYSL ω-Aga-IIIA- 962 FGYLKSGCKC VVGTSAEFQG ICRRKARTCY T58 NSDPDKCESH NKPKRR SCIDFGGDCD GEKDDCQCCR SNGYCSCYSL ω-Aga-IIIB 963 FGYLKSGCKC EVGTSAEFRR ICRRKAKQCY NSDPDKCVSV YKPKRR SCIDFGGDCD GEKDDCQCCR SNGYCSCYNL ω-Aga-IIIB- 964 FGYLKSGCKC EVGTSAEFRR N29 ICRRKAKQCYNSDPDKCVSV YKPKRR SCIDFGGDCD GEKDDCQCCR SNGYCSCYNL ω-Aga-IIIB- 965 FGYLRSGCKC EVGTSAEFRR ICRRKAKQCY N29/R35 NSDPDKCVSV YKPKRR NCIDFGGDCD GEKDDCQCCX RNGYCSCYNL ω-Aga-IIIC 966 FGYLKRGCKX EVG SCIKIGEDCD GDKDDCQCCR TNGYCSXYXL ω-Aga-IIID 967 FGYLKSG GCIEIGGDCD GYQEKSYCQC CRNNGFCS ω-Aga-IIA 968 AKAL PPGSVCDGNE SDCKCYGKWH ω-Aga-IA 969 KCRCPWKWHF TGEGPCTCEK GMKHTCITKL (major HCPNKAEWGL DW chain) ECVPENGHCR DWYDECCEGF YCSCRQPPKC μ-Aga 970 ICRNNNX DCVGESQQCA DWAGPHCCDG YYCTCRYFPK μ-Aga-6 971 CICVNNN ACVGENKQCA DWAGPHCCDG YYCTCRYFPK μ-Aga-5 972 CICRNNN ACVGENQQCA DWAGPHCCDG YYCTCRYFPK μ-Aga-4 973 CICRNNN ADCVGDGQRC ADWAGPYCCS GYYCSCRSMP μ-Aga-3 1275 YCRCRSDS ECATKNKRCA DWAGPWCCDG LYCSCRSYPG μ-Aga-2 974 CMCRPSS ECVPENGHCR DWYDECCEGF YCSCRQPPKC μ-Aga-1 975 ICRNNN AELTSCFPVGHECDGDASNCNCCGDDVYCGC Tx-1 1277 GWGRWNCKCKVADQSYAYGICKDKVNCPNRHL WPAKVCKKPCRREC GCANAYKSCNGPHTCCWGYNGYKKACICSGX Tx3-3 1278 NWK SCINVGDFCDGKKDCCQCDRDNAFCSCSVIFG Tx3-4 1279 YKTNCRCE SCINVGDFCDGKKDDCQCCRDNAFCSCSVI ω-PtXIIA 1280 FGYKTNCRCEVGTTATSYGICMAKHKCGRQT TCTKPCLSKRCKKNH AECLMIGDTSCVPRLGRRCCYGAWCYCDQQL Dw13.3 1281 SCRRVGRKRECGWVEVNCKCGWSWSQRID DWRADYSCKCPEDQ GGCLPHNRFCNALSGPRCCSGLKCKELSIW Agelenin 1282 DSRCL DCVRFWGKCSQTSDCCPHLACKSKWP ω-GTx-SIA 1283 RNICVWDGSV GCLEVDYFCG IPFANNGLCC SGNCVF ω-conotoxin 1284 VCTP Q PnVIA DDDCEPPGNF CGMIKIGPPC CSGWCFFACA ω-conotoxin 1285 PnVIB VCCGYKLCHP C Lambda- 1286 conotoxin CMrVIA MRCLPVLIIL LLLTASAPGV VVLPKTEDDV Lambda- 1287 PMSSVYGNGK SILRGILRNG conotoxin VCCGYKLCHP C CMrVIB KIDGYPVDYW NCKRICWYNN KYCNDLCKGL Kurtoxin 1276 KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA CKGKGAPCRKTMYDCCSGSCGRRGKC MVIIC 1368

In accordance with this invention are molecules in which at least one of the toxin peptide (P) portions of the molecule comprises a Kv1.3 antagonist peptide. Amino acid sequences selected from ShK, HmK, MgTx, AgTx1, AgTx2, Heterometrus spinnifer (HsTx1), OSK1, Anuroctoxin (AnTx), Noxiustoxin (NTX), KTX1, Hongotoxin, ChTx, Titystoxin, BgK, BmKTX, BmTx, AeK, AsKS Tc30, Tc32, Pi1, Pi2, and/or Pi3 toxin peptides and peptide analogs of any of these are preferred. Examples of useful Kv1.3 antagonist peptide sequences include those having any amino acid sequence set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, and/or Table 11 herein above;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is an IKCa1 antagonist peptide. Useful IKCa1 antagonist peptides include Maurotoxin (MTx), ChTx, peptides and peptide analogs of either of these, examples of which include those having any amino acid sequence set forth in Table 12, Table 13, and/or Table 14;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a SKCa inhibitor peptide. Useful SKCa inhibitor peptides include, Apamin, ScyTx, BmP05, P01, P05, Tamapin, TsK, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 15;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is an apamin peptide, and peptide analogs of apamin, examples of which include those having any amino acid sequence set forth in Table 16;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Scyllotoxin family peptide, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 17;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a BKCa inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 18;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Slotoxin family peptide, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 19;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Martentoxin peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 20;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a N-type Ca²⁺ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 21;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ωMVIIA peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 22;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ωGVIA peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 23;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Ptu1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 24;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ProTx1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 25;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a BeKM1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 26;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Na⁺ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 27;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Cl⁻ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 28;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Kv2.1 inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 29;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Kv4.2/Kv4.3 inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 30;

Other embodiments of the inventive composition include at least one toxin peptide (P) that is a nACHR inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 31; and

Other embodiments of the inventive composition include at least one toxin peptide (P) that is an Agatoxin peptide, a peptide analog thereof or other calcium channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 32.

Half-life extending moieties. This invention involves the presence of at least one half-life extending moiety (F¹ and/or F² in Formula I) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the intracalary amino acid residues. Multiple half-life extending moieties can also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or at a sidechain. In other embodiments the Fc domain can be PEGylated (e.g., in accordance with the formulae F¹—F²-(L)_(f)-P; P-(L)_(g)-F¹—F²; or P-(L)₉-F¹—F²-(L)_(f)-P).

The half-life extending moiety can be selected such that the inventive composition achieves a sufficient hydrodynamic size to prevent clearance by renal filtration in vivo. For example, a half-life extending moiety can be selected that is a polymeric macromolecule, which is substantially straight chain, branched-chain, or dendritic in form. Alternatively, a half-life extending moiety can be selected such that, in vivo, the inventive composition of matter will bind to a serum protein to form a complex, such that the complex thus formed avoids substantial renal clearance. The half-life extending moiety can be, for example, a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.

Exemplary half-life extending moieties that can be used, in accordance with the present invention, include an immunoglobulin Fc domain, or a portion thereof, or a biologically suitable polymer or copolymer, for example, a polyalkylene glycol compound, such as a polyethylene glycol or a polypropylene glycol. Other appropriate polyalkylene glycol compounds include, but are not limited to, charged or neutral polymers of the following types: dextran, polylysine, colominic acids or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives. In some monomeric fusion protein embodiments an immunoglobulin (including light and heavy chains) or a portion thereof, can be used as a half-life-extending moiety, preferably an immunoglobulin of human origin, and including any of the immunoglobulins, such as, but not limited to, IgG1, IgG2, IgG3 or IgG4.

Other examples of the half-life extending moiety, in accordance with the invention, include a copolymer of ethylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin light chain and heavy chain, an immunoglobulin Fc domain or a portion thereof (see, e.g., Feige et al., Modified peptides as therapeutic agents, U.S. Pat. No. 6,660,843), a CH2 domain of Fc, an albumin (e.g., human serum albumin (HSA)); see, e.g., Rosen et al., Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US 2005/0054051; Bridon et al., Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components, U.S. Pat. No. 6,887,470), a transthyretin (TTR; see, e.g., Walker et al., Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins, US 2003/0195154 A1; 2003/0191056 A1), or a thyroxine-binding globulin (TBG). Thus, exemplary embodiments of the inventive compositions also include HSA fusion constructs such as but not limited to: HSA fusions with ShK, OSK1, or modified analogs of those toxin peptides. Examples include HSA-L10-ShK(2-35); HSA-L10-OsK1(1-38); HSA-L10-ShK(2-35); and HSA-L10-OsK1(1-38).

Other embodiments of the half-life extending moiety, in accordance with the invention, include peptide ligands or small (organic) molecule ligands that have binding affinity for a long half-life serum protein under physiological conditions of temperature, pH, and ionic strength. Examples include an albumin-binding peptide or small molecule ligand, a transthyretin-binding peptide or small molecule ligand, a thyroxine-binding globulin-binding peptide or small molecule ligand, an antibody-binding peptide or small molecule ligand, or another peptide or small molecule that has an affinity for a long half-life serum protein. (See, e.g., Blaney et al., Method and compositions for increasing the serum half-life of pharmacologically active agents by binding to transthyretin-selective ligands, U.S. Pat. No. 5,714,142; Sato et al., Serum albumin binding moieties, US 2003/0069395 A1; Jones et al., Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225). A “long half-life serum protein” is one of the hundreds of different proteins dissolved in mammalian blood plasma, including so-called “carrier proteins” (such as albumin, transferrin and haptoglobin), fibrinogen and other blood coagulation factors, complement components, immunoglobulins, enzyme inhibitors, precursors of substances such as angiotensin and bradykinin and many other types of proteins. The invention encompasses the use of any single species of pharmaceutically acceptable half-life extending moiety, such as, but not limited to, those described herein, or the use of a combination of two or more different half-life extending moieties, such as PEG and immunoglobulin Fc domain or a CH2 domain of Fc, albumin (e.g., HSA), an albumin-binding protein, transthyretin or TBG, or a combination such as immunoglobulin (light chain+heavy chain) and Fc domain (the combination so-called “hemibody”).

In some embodiments of the invention an Fc domain or portion thereof, such as a CH2 domain of Fc, is used as a half-life extending moiety. The Fc domain can be fused to the N-terminal (e.g., in accordance with the formula F¹-(L)_(f)-P) or C-terminal (e.g., in accordance with the formula P-(L)_(g)-F¹) of the toxin peptides or at both the N and C termini (e.g., in accordance with the formulae F¹-(L)_(f)-P-(L)_(g)-F² or P-(L)_(g)-F¹-(L)_(f)-P). A peptide linker sequence can be optionally included between the Fc domain and the toxin peptide, as described herein. Examples of the formula F¹-(L)_(f)-P include: Fc-L10-ShK(K22A)[2-35]; Fc-L10-ShK(R1K/K22A)[1-35]; Fc-L10-ShK(R1H/K22A)[1-35]; Fc-L10-ShK(R1Q/K22A)[1-35]; Fc-L10-ShK(R1Y/K22A)[1-35]; Fc-L10-PP-ShK(K22A) [1-35]; and any other working examples described herein. Examples of the formula P-(L)_(g)-F¹ include: ShK(1-35)-L10-Fc; OsK1(1-38)-L10-Fc; Met-ShK(1-35)-L10-Fc; ShK(2-35)-L10-Fc; Gly-ShK(1-35)-L10-Fc; Osk1(1-38)-L10-Fc; and any other working examples described herein.

Fc variants are suitable half-life extending moieties within the scope of this invention. A native Fc can be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631, WO 96/32478, and WO 04/110 472. In such Fc variants, one can remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One can remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues can also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants can be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

-   -   1. Sites involved in disulfide bond formation are removed. Such         removal can avoid reaction with other cysteine-containing         proteins present in the host cell used to produce the molecules         of the invention. For this purpose, the cysteine-containing         segment at the N-terminus can be truncated or cysteine residues         can be deleted or substituted with other amino acids (e.g.,         alanyl, seryl). In particular, one can truncate the N-terminal         20-amino acid segment of SEQ ID NO: 2 or delete or substitute         the cysteine residues at positions 7 and 10 of SEQ ID NO: 2.         Even when cysteine residues are removed, the single chain Fc         domains can still form a dimeric Fc domain that is held together         non-covalently.     -   2. A native Fc is modified to make it more compatible with a         selected host cell. For example, one can remove the PA sequence         near the N-terminus of a typical native Fc, which can be         recognized by a digestive enzyme in E. coli such as proline         iminopeptidase. One can also add an N-terminal methionine         residue, especially when the molecule is expressed recombinantly         in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO:         2 (FIG. 4A-4B) is one such Fc variant.     -   3. A portion of the N-terminus of a native Fc is removed to         prevent N-terminal heterogeneity when expressed in a selected         host cell. For this purpose, one can delete any of the first 20         amino acid residues at the N-terminus, particularly those at         positions 1, 2, 3, 4 and 5.     -   4. One or more glycosylation sites are removed. Residues that         are typically glycosylated (e.g., asparagine) can confer         cytolytic response. Such residues can be deleted or substituted         with unglycosylated residues (e.g., alanine).     -   5. Sites involved in interaction with complement, such as the         C1q binding site, are removed. For example, one can delete or         substitute the EKK sequence of human IgG1. Complement         recruitment may not be advantageous for the molecules of this         invention and so can be avoided with such an Fc variant.     -   6. Sites are removed that affect binding to Fc receptors other         than a salvage receptor. A native Fc can have sites for         interaction with certain white blood cells that are not required         for the fusion molecules of the present invention and so can be         removed.     -   7. The ADCC site is removed. ADCC sites are known in the art;         see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with         regard to ADCC sites in IgG1. These sites, as well, are not         required for the fusion molecules of the present invention and         so can be removed.     -   8. When the native Fc is derived from a non-human antibody, the         native Fc can be humanized. Typically, to humanize a native Fc,         one will substitute selected residues in the non-human native Fc         with residues that are normally found in human native Fc.         Techniques for antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2, the leucine at position 15 can be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, phenyalanine residues can replace one or more tyrosine residues.

An alternative half-life extending moiety would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a half-life extending moiety a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “half-life extending moiety” and are within the scope of this invention. Such half-life extending moieties should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).

As noted above, polymer half-life extending moieties can also be used for F¹ and F². Various means for attaching chemical moieties useful as half-life extending moieties are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water-soluble polymers to the N-terminus of proteins.

In some embodiments of the inventive compositions, the polymer half-life extending moiety is polyethylene glycol (PEG), as F¹ and/or F², but it should be understood that the inventive composition of matter, beyond positions F¹ and/or F², can also include one or more PEGs conjugated at other sites in the molecule, such as at one or more sites on the toxin peptide. Accordingly, some embodiments of the inventive composition of matter further include one or more PEG moieties conjugated to a non-PEG half-life extending moiety, which is F¹ and/or F², or to the toxin peptide(s) (P), or to any combination of any of these. For example, an Fc domain or portion thereof (as F1 and/or F2) in the inventive composition can be made mono-PEGylated, di-PEGylated, or otherwise multi-PEGylated, by the process of reductive alkylation.

Covalent conjugation of proteins and peptides with poly(ethylene glycol) (PEG) has been widely recognized as an approach to significantly extend the in vivo circulating half-lives of therapeutic proteins. PEGylation achieves this effect predominately by retarding renal clearance, since the PEG moiety adds considerable hydrodynamic radius to the protein. (Zalipsky, S., et al., Use of functionalized poly(ethylene glycol)s for modification of polypeptides., in poly(ethylene glycol) chemistry: Biotechnical and biomedical applications., J. M. Harris, Ed., Plenum Press: New York., 347-370 (1992)). Additional benefits often conferred by PEGylation of proteins and peptides include increased solubility, resistance to proteolytic degradation, and reduced immunogenicity of the therapeutic polypeptide. The merits of protein PEGylation are evidenced by the commercialization of several PEGylated proteins including PEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase (Oncaspar™/Enzon Corp.), PEG-Interferon α-2b (PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) and PEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.

Briefly, the PEG groups are generally attached to the peptide portion of the composition of the invention via acylation or reductive alkylation (or reductive amination) through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).

A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). In the present application, the term “PEG” is used broadly to encompass any polyethylene glycol molecule, in mono-, bi-, or poly-functional form, without regard to size or to modification at an end of the PEG, and can be represented by the formula: X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,  (X)

where n is 20 to 2300 and X is H or a terminal modification, e.g., a C₁₋₄ alkyl.

In some useful embodiments, a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). It is noted that the other end of the PEG, which is shown in formula (II) terminating in OH, covalently attaches to an activating moiety via an ether oxygen bond, an amine linkage, or amide linkage. When used in a chemical structure, the term “PEG” includes the formula (II) above without the hydrogen of the hydroxyl group shown, leaving the oxygen available to react with a free carbon atom of a linker to form an ether bond. More specifically, in order to conjugate PEG to a peptide, the peptide must be reacted with PEG in an “activated” form. Activated PEG can be represented by the formula: (PEG)-(A)  (XI) where PEG (defined supra) covalently attaches to a carbon atom of the activation moiety (A) to form an ether bond, an amine linkage, or amide linkage, and (A) contains a reactive group which can react with an amino, azido, alkyne, imino, maleimido, N-succinimidyl, carboxyl, aminooxy, seleno, or thiol group on an amino acid residue of a peptide or a linker moiety covalently attached to the peptide, e.g., the OSK1 peptide analog. Residues baring chemoselective reactive groups can be introduced into the toxin peptide, e.g., an OSK1 peptide analog during assembly of the peptide sequence solid-phase synthesis as protected derivatives. Alternatively, chemoselective reactive groups can be introduced in the toxin peptide after assembly of the peptide sequence by solid-phase synthesis via the use of orthogonal protecting groups at specific sites. Examples of amino acid residues useful for chemoselective reactions include, but are not limited to, (amino-oxyacetyl)-L-diaminopropionic acid, p-azido-phenylalanine, azidohomolalanine, para-propargyloxy-phenylalanine, selenocysteine, para-acetylphenylalanine, (N^(ε)-levulinyl)-Lysine, (N^(ε)-pyruvyl)-Lysine, selenocysteine, and orthogonally protected cysteine and homocysteine.

Accordingly, in some embodiments of the composition of matter, the toxin peptide, e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol (PEG) at:

(a) 1, 2, 3 or 4 amino functionalized sites in the toxin peptide;

(b) 1, 2, 3 or 4 thiol functionalized sites in the toxin peptide; (c) 1 or 2 ketone functionalized sites in the toxin peptide; (d) 1 or 2 azido functionalized sites of the toxin peptide; (e) 1 or 2 carboxyl functionalized sites in the toxin peptide; (f) 1 or 2 aminooxy functionalized sites in the toxin peptide; or (g) 1 or 2 seleno functionalized sites in the toxin peptide.

In other embodiments of the composition of matter, the toxin peptide, e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol (PEG) at:

(a) 1, 2, 3 or 4 amino functionalized sites of the PEG;

(b) 1, 2, 3 or 4 thiol functionalized sites of the PEG;

(c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG;

(d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG;

(e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG; or

(f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of the PEG.

Techniques for the preparation of activated PEG and its conjugation to biologically active peptides are well known in the art. (E.g., see U.S. Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237; Thompson et al., PEGylation of polypeptides, EP 0575545 B1; Petit, Site specific protein modification, U.S. Pat. Nos. 6,451,986, and 6,548,644; S. Herman et al., Poly(ethylene glycol) with reactive endgroups: I. Modification of proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); Y. Lu et al., Pegylated peptides III: Solid-phase synthesis with PEGylating reagents of varying molecular weight: synthesis of multiply PEGylated peptides, Reactive Polymers, 22:221-229 (1994); A. M. Felix et al., PEGylated Peptides IV: Enhanced biological activity of site-directed PEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264 (1995); A. M. Felix, Site-specific poly(ethylene glycol)ylation of peptides, ACS Symposium Series 680(poly(ethylene glycol)): 218-238 (1997); Y. Ikeda et al., Polyethylene glycol derivatives, their modified peptides, methods for producing them and use of the modified peptides, EP 0473084 B1; G. E. Means et al., Selected techniques for the modification of protein side chains, in: Chemical modification of proteins, Holden Day, Inc., 219 (1971)).

Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can be chemically synthesized by known means or obtained from commercial sources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J.).

An example of a useful activated PEG for purposes of the present invention is a PEG-aldehyde compound (e.g., a methoxy PEG-aldehyde), such as PEG-propionaldehyde, which is commercially available from Shearwater Polymers (Huntsville, Ala.). PEG-propionaldehyde is represented by the formula PEG-CH₂CH₂CHO. (See, e.g., U.S. Pat. No. 5,252,714). Other examples of useful activated PEG are PEG acetaldehyde hydrate and PEG bis aldehyde hydrate, which latter yields a bifunctionally activated structure. (See., e.g., Bentley et al., Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines, U.S. Pat. No. 5,990,237).

Another useful activated PEG for generating the PEG-conjugated peptides of the present invention is a PEG-maleimide compound, such as, but not limited to, a methoxy PEG-maleimide, such as maleimido monomethoxy PEG, are particularly useful for generating the PEG-conjugated peptides of the invention. (E.g., Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498; C. Delgado et al., The uses and properties of PEG-linked proteins., Crit. Rev. Therap. Drug Carrier Systems, 9:249-304 (1992); S. Zalipsky et al., Use of functionalized poly(ethylene glycol)s for modification of polypeptides, in: Poly(ethylene glycol) chemistry: Biotechnical and biomedical applications (J. M. Harris, Editor, Plenum Press: New York, 347-370 (1992); S. Herman et al., Poly(ethylene glycol) with reactive endgroups: I. Modification of proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); P. J. Shadle et al., Conjugation of polymer to colony stimulating factor-1, U.S. Pat. No. 4,847,325; G. Shaw et al., Cysteine added variants IL-3 and chemical modifications thereof, U.S. Pat. No. 5,166,322 and EP 0469074 B1; G. Shaw et al., Cysteine added variants of EPO and chemical modifications thereof, EP 0668353 A1; G. Shaw et al., Cysteine added variants G-CSF and chemical modifications thereof, EP 0668354 A1; N. V. Katre et al., Interleukin-2 muteins and polymer conjugation thereof, U.S. Pat. No. 5,206,344; R. J. Goodson and N. V. Katre, Site-directed pegylation of recombinant interleukin-2 at its glycosylation site, Biotechnology, 8:343-346 (1990)).

A poly(ethylene glycol) vinyl sulfone is another useful activated PEG for generating the PEG-conjugated peptides of the present invention by conjugation at thiolated amino acid residues, e.g., at C residues. (E.g., M. Morpurgo et al., Preparation and characterization of poly(ethylene glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996); see also Harris, Functionalization of polyethylene glycol for formation of active sulfone-terminated PEG derivatives for binding to proteins and biologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208; 5,900,461; 6,610,281 and 6,894,025; and Harris, Water soluble active sulfones of poly(ethylene glycol), WO 95/13312 A1).

Another activated form of PEG that is useful in accordance with the present invention, is a PEG-N-hydroxysuccinimide ester compound, for example, methoxy PEG-N-hydroxysuccinimidyl (NHS) ester.

Heterobifunctionally activated forms of PEG are also useful. (See, e.g., Thompson et al., PEGylation reagents and biologically active compounds formed therewith, U.S. Pat. No. 6,552,170).

Typically, a toxin peptide or, a fusion protein comprising the toxin peptide, is reacted by known chemical techniques with an activated PEG compound, such as but not limited to, a thiol-activated PEG compound, a diol-activated PEG compound, a PEG-hydrazide compound, a PEG-oxyamine compound, or a PEG-bromoacetyl compound. (See, e.g., S. Herman, Poly(ethylene glycol) with Reactive Endgroups: I. Modification of Proteins, J. Bioactive and Compatible Polymers, 10:145-187 (1995); S. Zalipsky, Chemistry of Polyethylene Glycol Conjugates with Biologically Active Molecules, Advanced Drug Delivery Reviews, 16:157-182 (1995); R. Greenwald et al., Poly(ethylene glycol) conjugated drugs and prodrugs: a comprehensive review, Critical Reviews in Therapeutic Drug Carrier Systems, 17:101-161 (2000)).

Methods for N-terminal PEGylation are exemplified herein in Examples 31-34, 45 and 47-48, but these are in no way limiting of the PEGylation methods that can be employed by one skilled in the art.

Any molecular mass for a PEG can be used as practically desired, e.g., from about 1,000 or 2,000 Daltons (Da) to about 100,000 Da (n is 20 to 2300). Preferably, the combined or total molecular mass of PEG used in a PEG-conjugated peptide of the present invention is from about 3,000 Da or 5,000 Da, to about 50,000 Da or 60,000 Da (total n is from 70 to 1,400), more preferably from about 10,000 Da to about 40,000 Da (total n is about 230 to about 910). The most preferred combined mass for PEG is from about 20,000 Da to about 30,000 Da (total n is about 450 to about 680). The number of repeating units “n” in the PEG is approximated for the molecular mass described in Daltons. It is preferred that the combined molecular mass of PEG on an activated linker is suitable for pharmaceutical use. Thus, the combined molecular mass of the PEG molecule should not exceed about 100,000 Da.

Polysaccharide polymers are another type of water-soluble polymer that can be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa. Dextran is a suitable water-soluble polymer for use in the present invention as a half-life extending moiety by itself or in combination with another half-life extending moiety (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference in its entirety. Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a half-life extending moiety in accordance with the present invention.

Linkers. Any “linker” group or moiety (i.e., “-(L)_(g)-” or “-(L)_(g)-” in Formulae I-IX) is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. As stated herein above, the linker moiety (-(L)_(f)- and/or -(L)_(g)-), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. For example, an “(L)_(f)” can represent the same moiety as, or a different moiety from, any other “(L)_(f)” or any “(L)_(g)” in accordance with the invention. The linker is preferably made up of amino acids linked together by peptide bonds. Some of these amino acids can be glycosylated, as is well understood by those in the art. For example, a useful linker sequence constituting a sialylation site is X₁X₂NX₄X₅G (SEQ ID NO: 637), wherein X₁, X₂, X₄ and X₅ are each independently any amino acid residue.

As stated above, in some embodiments, a peptidyl linker is present (i.e., made up of amino acids linked together by peptide bonds) that is made in length, preferably, of from 1 up to about 40 amino acid residues, more preferably, of from 1 up to about 20 amino acid residues, and most preferably of from 1 to about 10 amino acid residues. Preferably, but not necessarily, the amino acid residues in the linker are from among the twenty canonical amino acids, more preferably, cysteine, glycine, alanine, proline, asparagine, glutamine, and/or serine. Even more preferably, a peptidyl linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, serine, and alanine linked by a peptide bond. It is also desirable that, if present, a peptidyl linker be selected that avoids rapid proteolytic turnover in circulation in vivo. Thus, preferred linkers include polyglycines (particularly (Gly)₄ (SEQ ID NO: 4918), (Gly)₅) (SEQ ID NO: 4919), poly(Gly-Ala), and polyalanines. Other preferred linkers are those identified herein as “L5” (GGGGS; SEQ ID NO: 638), “L10” (GGGGSGGGGS; SEQ ID NO:79), “L25” GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO:84) and any linkers used in the working examples hereinafter. The linkers described herein, however, are exemplary; linkers within the scope of this invention can be much longer and can include other residues.

In some embodiments of the compositions of this invention, which comprise a peptide linker moiety (L), acidic residues, for example, glutamate or aspartate residues, are placed in the amino acid sequence of the linker moiety (L). Examples include the following peptide linker sequences:

GGEGGG; (SEQ ID NO: 639) GGEEEGGG; (SEQ ID NO: 640) GEEEG; (SEQ ID NO: 641) GEEE; (SEQ ID NO: 642) GGDGGG; (SEQ ID NO: 643) GGDDDGG; (SEQ ID NO: 644) GDDDG; (SEQ ID NO: 645) GDDD; (SEQ ID NO: 646) GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 647) WEWEW; (SEQ ID NO: 648) FEFEF; (SEQ ID NO: 649) EEEWWW; (SEQ ID NO: 650) EEEFFF; (SEQ ID NO: 651) WWEEEWW; (SEQ ID NO: 652) or FFEEEFF. (SEQ ID NO: 653)

In other embodiments, the linker constitutes a phosphorylation site, e.g., X₁X₂YX₃X₄G (SEQ ID NO: 654), wherein X₁, X₂, X₃ and X₄ are each independently any amino acid residue; X₁X₂SX₃X₄G (SEQ ID NO: 655), wherein X₁, X₂, X₃ and X₄ are each independently any amino acid residue; or X₁X₂TX₃X₄G (SEQ ID NO: 656), wherein X₁, X₂, X₃ and X₄ are each independently any amino acid residue.

Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyl linkers can further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000 kDa, preferably 100 to 500 kDa. The peptide linkers can be altered to form derivatives in the same manner as described above.

Useful linker embodiments also include aminoethyloxyethyloxy-acetyl linkers as disclosed by Chandy et al. (Chandy et al., WO 2006/042151 A2, incorporated herein by reference in its entirety).

Derivatives. The inventors also contemplate derivatizing the peptide and/or half-life extending moiety portion of the compounds. Such derivatives can improve the solubility, absorption, biological half-life, and the like of the compounds. The moieties can alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which:

-   1. The compound or some portion thereof is cyclic. For example, the     peptide portion can be modified to contain two or more Cys residues     (e.g., in the linker), which could cyclize by disulfide bond     formation. -   2. The compound is cross-linked or is rendered capable of     cross-linking between molecules. For example, the peptide portion     can be modified to contain one Cys residue and thereby be able to     form an intermolecular disulfide bond with a like molecule. The     compound can also be cross-linked through its C-terminus, as in the     molecule shown below.

-   3. Non-peptidyl linkages (bonds) replace one or more peptidyl     [—C(O)NR—] linkages. Exemplary non-peptidyl linkages are     —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide     [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and     alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl]. -   4. The N-terminus is chemically derivatized. Typically, the     N-terminus can be acylated or modified to a substituted amine.     Exemplary N-terminal derivative groups include —NRR¹ (other than     —NH₂), —NRC(O)R¹, -    —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or     benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R¹ are each     independently hydrogen or lower alkyl and wherein the phenyl ring     can be substituted with 1 to 3 substituents selected from the group     consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo. -   5. The free C-terminus is derivatized. Typically, the C-terminus is     esterified or amidated. For example, one can use methods described     in the art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention     having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise,     one can use methods described in the art to add —NH₂ to compounds of     this invention having any of SEQ ID NOS: 924 to 955, 963 to 972,     1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary     C-terminal derivative groups include, for example, —C(O)R² wherein     R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently     hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl). -   6. A disulfide bond is replaced with another, preferably more     stable, cross-linking moiety (e.g., an alkylene). See, e.g.,     Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et     al. (1993) Thirteenth Am. Pep. Symp., 357-9. -   7. One or more individual amino acid residues are modified. Various     derivatizing agents are known to react specifically with selected     sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues can be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues can be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with the groups of lysine as well as the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues can be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular half-life extending moieties. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups can conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, can confer acidic properties to the glycosylated compound. Such site(s) can be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites can further be glycosylated by synthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman and Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention can be changed at the DNA level, as well. The DNA sequence of any portion of the compound can be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which can aid in processing of the DNA in the selected host cell. The half-life extending moiety, linker and peptide DNA sequences can be modified to include any of the foregoing sequence changes.

A process for preparing conjugation derivatives is also contemplated. Tumor cells, for example, exhibit epitopes not found on their normal counterparts. Such epitopes include, for example, different post-translational modifications resulting from their rapid proliferation. Thus, one aspect of this invention is a process comprising:

-   -   a) selecting at least one randomized peptide that specifically         binds to a target epitope; and     -   b) preparing a pharmacologic agent comprising (i) at least one         half-life extending moiety (Fc domain preferred), (ii) at least         one amino acid sequence of the selected peptide or peptides,         and (iii) an effector molecule.         The target epitope is preferably a tumor-specific epitope or an         epitope specific to a pathogenic organism. The effector molecule         can be any of the above-noted conjugation partners and is         preferably a radioisotope.

Methods of Making

The present invention also relates to nucleic acids, expression vectors and host cells useful in producing the polypeptides of the present invention. Host cells can be eukaryotic cells, with mammalian cells preferred and CHO cells most preferred. Host cells can also be prokaryotic cells, with E. coli cells most preferred.

The compounds of this invention largely can be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation can be performed using methods well known in the art.

Any of a large number of available and well-known host cells can be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts can be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

Next, the transformed host is cultured and purified. Host cells can be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.

In some embodiments of the inventive DNA, the DNA encodes a recombinant fusion protein composition of the invention, preferably, but not necessarily, monovalent with respect to the toxin peptide, for expression in a mammalian cell, such as, but not limited to, CHO or HEK293. The encoded fusion protein includes (a)-(c) immediately below, in the N-terminal to C-terminal direction:

(a) an immunoglobulin, which includes the constant and variable regions of the immunoglobulin light and heavy chains, or a portion of an immunoglobulin (e.g., an Fc domain, or the variable regions of the light and heavy chains); if both immunoglobulin light chain and heavy chain components are to be included in the construct, then a peptidyl linker, as further described in (b) immediately below, is also included to separate the immunoglobulin components (See, e.g., FIG. 92A-C); useful coding sequences for immunoglobulin light and heavy chains are well known in the art;

(b) a peptidyl linker, which is at least 4 (or 5) amino acid residues long and comprises at least one protease cleavage site (e.g., a furin cleavage site, which is particularly useful for intracellular cleavage of the expressed fusion protein); typically, the peptidyl linker sequence can be up to about 35 to 45 amino acid residues long (e.g., a 7× L5 linker modified to include the desired protease cleavage site(s)), but linkers up to about 100 to about 300 amino acid residues long are also useful; and

(c) an immunoglobulin Fc domain or a portion thereof. The Fc domain of (c) can be from the same type of immunoglobulin in (a), or different. In such embodiments, the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c) above, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site). Any toxin peptide or peptide analog thereof as described herein can be encoded by the DNA (e.g., but not limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin peptide, or a peptide analog of any of these). For example, an OSK1 peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015, as set forth in Tables 7 and Tables 7A-J, can be employed. Alternatively, an ShK peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, and 1295 through 1300 as set forth in Table 2, can be employed. Any other toxin peptide sequence described herein that can alternatively be expressed recombinantly using recombinant and protein engineering techniques known in the art can also be used. The immunoglobulin of (a) and (c) above can be in each instance independently selected from any desired type, such as but not limited to, IgG1, IgG2, IgG3, and IgG4. The variable regions can be non-functional in vivo (e.g., CDRs specifically binding KLH), or alternatively, if targeting enhancement function is also desired, the variable regions can be chosen to specifically bind (non-competitively) the ion channel target of the toxin peptide (e.g., Kv1.3) or specifically bind another antigen typically found associated with, or in the vicinity of, the target ion channel. In addition, the inventive DNA optionally further encodes, 5′ to the coding region of (a) above, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed fusion protein. An example of the inventive DNA encoding a recombinant fusion protein for expression in a mammalian cell, described immediately above, is a DNA that encodes a fusion protein comprising, in the N-terminal to C-terminal direction:

(a) an immunoglobulin light chain;

(b) a first peptidyl linker at least 4 amino acid residues long comprising at least one protease cleavage site, as described above;

(c) an immunoglobulin heavy chain;

(d) a second peptidyl linker at least 4 amino acid residues long comprising at least one protease cleavage site, as described above; and

(e) an immunoglobulin Fc domain or a portion thereof. Here, the Fc domain of (e) can be from the same type of immunoglobulin as the heavy chain in (c), or different. The DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a), (c), or (e) of the expressed fusion protein, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site). FIG. 92A-C illustrates schematically an embodiment, in which the toxin peptide (e.g., an OSK1, ShK, or a peptide analog of either of these) is covalently linked to the C-terminal end of the Fc domain of (e). In FIG. 92A-C, a linker is shown covalently linking the toxin peptide to the rest of the molecule, but as previously described, this linker is optional.

In some embodiments particularly suited for the recombinant expression of monovalent dimeric Fc-toxin peptide fusions or “peptibodies” (see, FIG. 2B and Example 56) by mammalian cells, such as, but not limited to, CHO or HEK293, the inventive DNA encodes a recombinant expressed fusion protein that comprises, in the N-terminal to C-terminal direction:

-   -   (a) a first immunoglobulin Fc domain or portion thereof;     -   (b) a peptidyl linker at least 4 (or 5) amino acid residues long         comprising at least one protease cleavage site (e.g., a furin         cleavage site, which is particularly useful for intracellular         cleavage of the expressed fusion protein); typically, the         peptidyl linker sequence can be up to about 35 to 45 amino acid         residues long (e.g., a 7×L5 linker modified to include the         desired protease cleavage site(s)), but linkers up to about 100         to about 300 amino acid residues long are also useful; and     -   (c) a second immunoglobulin Fc domain or portion thereof (which         may be the same or different from the first Fc domain, but         should be expressed in the same orientation as the first Fc         domain).         For such embodiments, the DNA encodes a toxin peptide covalently         linked to the N-terminal or C-terminal end of (a) or (c) of the         expressed fusion protein, either directly or indirectly via a         peptidyl linker (a linker minus a protease cleavage site);         Example 56 describes an embodiment in which the toxin peptide is         conjugated to the C-terminal end of the second immunoglobulin Fc         domain (c). Any toxin peptide or peptide analog thereof as         described herein can be encoded by the DNA (e.g., but not         limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1,         Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx,         Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin         peptide, or a peptide analog of any of these). For example, an         OSK1 peptide analog comprising an amino acid sequence selected         from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980         through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through         5006, 5009, 5010, and 5012 through 5015, as set forth in Tables         7 and Tables 7A-J, can be employed. Alternatively, an ShK         peptide analog comprising an amino acid sequence selected from         SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950,         and 1295 through 1300 as set forth in Table 2, can be employed.         Any other toxin peptide sequence described herein that can         alternatively be expressed using recombinant and protein         engineering techniques known in the art can also be used. In         addition, the inventive DNA optionally further encodes, 5′ to         the coding region of (a) above, a signal peptide sequence (e.g.,         a secretory signal peptide) operably linked to the expressed         fusion protein.

DNA constructs similar to those described above are also useful for recombinant expression by mammalian cells of other dimeric Fc fusion proteins (“peptibodies”) or chimeric immunoglobulin (light chain+heavy chain)-Fc heterotrimers (“hemibodies”), conjugated to pharmacologically active peptides (e.g., agonist or antagonist peptides) other than toxin peptides.

Peptide compositions of the present invention can also be made by synthetic methods. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. For example, well known solid phase synthesis techniques include the use of protecting groups, linkers, and solid phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, use of scavengers, and other aspects of solid phase peptide synthesis. Suitable techniques are well known in the art. (E.g., Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527; “Protecting Groups in Organic Synthesis,” 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog, 2000; “Synthetic Peptides, A User's Guide,” G. A. Grant, Ed., W.H. Freeman & Company, New York, N.Y., 1992; “Advanced Chemtech Handbook of Combinatorial & Solid Phase Organic Chemistry,” W. D. Bennet, J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced Chemtech, 1998; “Principles of Peptide Synthesis, 2nd ed.,” M. Bodanszky, Ed., Springer-Verlag, 1993; “The Practice of Peptide Synthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields et al., Synthetic Peptides: A User's Guide, 1990, 77-183).

Whether the compositions of the present invention are prepared by synthetic or recombinant techniques, suitable protein purification techniques can also be involved, when applicable. In some embodiments of the compositions of the invention, the toxin peptide portion and/or the half-life extending portion, or any other portion, can be prepared to include a suitable isotopic label (e.g., ¹²⁵I, ¹⁴C, ¹³C, ³⁵S, ³H, ²H, ¹³N, ¹⁵N, ¹⁸O, ¹⁷O, etc.), for ease of quantification or detection.

Compounds that contain derivatized peptides or which contain non-peptide groups can be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

In general. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. Heritable diseases that have a known linkage to ion channels (“channelopathies”) cover various fields of medicine, some of which include neurology, nephrology, myology and cardiology. A list of inherited disorders attributed to ion channels includes:

-   -   cystic fibrosis (Cl⁻ channel; CFTR),     -   Dent's disease (proteinuria and hypercalciuria; Cl⁻ channel;         CLCN5),     -   osteopetrosis (Cl⁻ channel; CLCN7),     -   familial hyperinsulinemia (SUR1; KCNJ11; K channel),     -   diabetes (KATP/SUR channel),     -   Andersen syndrome (KCNJ2, Kir2.1 K channel),     -   Bartter syndrome (KCNJ1; Kir1.1/ROMK; K channel),     -   hereditary hearing loss (KCNQ4; K channel),     -   hereditary hypertension (Liddle's syndrome; SCNN1; epithelial Na         channel),     -   dilated cardiomyopathy (SUR2, K channel),     -   long-QT syndrome or cardiac arrhythmias (cardiac potassium and         sodium channels),     -   Thymothy syndrome (CACNA1C, Cav1.2),     -   myasthenic syndromes (CHRNA, CHRNB, CNRNE; nAChR), and a variety         of other myopathies,     -   hyperkalemic periodic paralysis (Na and K channels),     -   epilepsy (Na⁺ and K⁺ channels),     -   hemiplegic migraine (CACNA1A, Cav2.1 Ca²⁺ channel and ATP1A2),     -   central core disease (RYR1, RYR1; Ca²⁺ channel), and     -   paramyotonia and myotonia (Na⁺, Cl⁻ channels)         See L. J. Ptacek and Y-H Fu (2004), Arch. Neurol. 61:         166-8; B. A. Niemeyer et al. (2001), EMBO reports 21: 568-73; F.         Lehmann-Horn and K. Jurkat-Rott (1999), Physiol. Rev. 79:         1317-72. Although the foregoing list concerned disorders of         inherited origin, molecules targeting the channels cited in         these disorders can also be useful in treating related disorders         of other, or indeterminate, origin.

In addition to the aforementioned disorders, evidence has also been provided supporting ion channels as targets for treatment of:

-   -   sickle cell anemia (IKCa1)—in sickle cell anemia, water loss         from erythrocytes leads to hemoglobin polymerization and         subsequent hemolysis and vascular obstruction. The water loss is         consequent to potassium efflux through the so-called Gardos         channel i.e., IKCa1. Therefore, block of IKCa1 is a potential         therapeutic treatment for sickle cell anemia.     -   glaucoma (BKCa),—in glaucoma the intraocular pressure is too         high leading to optic nerve damage, abnormal eye function and         possibly blindness. Block of BKCa potassium channels can reduce         intraocular fluid secretion and increase smooth muscle         contraction, possibly leading to lower intraocular pressure and         neuroprotection in the eye.     -   multiple sclerosis (Kv, KCa),     -   psoriasis (Kv, KCa),     -   arthritis (Kv, KCa),     -   asthma (KCa, Kv),     -   allergy (KCa, Kv),     -   COPD (KCa, Kv, Ca),     -   allergic rhinitis (KCa, Kv),     -   pulmonary fibrosis,     -   lupus (IKCa1, Kv),     -   transplantation, GvHD (KCa, Kv),     -   inflammatory bone resorption (KCa, Kv),     -   periodontal disease (KCa, Kv),     -   diabetes, type I (Kv),—type I diabetes is an autoimmune disease         that is characterized by abnormal glucose, protein and lipid         metabolism and is associated with insulin deficiency or         resistance. In this disease, Kv1.3-expressing T-lymphocytes         attack and destroy pancreatic islets leading to loss of         beta-cells. Block of Kv1.3 decreases inflammatory cytokines. In         addition block of Kv1.3 facilitates the translocation of GLUT4         to the plasma membrane, thereby increasing insulin sensitivity.     -   obesity (Kv),—Kv1.3 appears to play a critical role in         controlling energy homeostasis and in protecting against         diet-induced obesity. Consequently, Kv1.3 blockers could         increase metabolic rate, leading to greater energy utilization         and decreased body weight.     -   restenosis (KCa, Ca²⁺),—proliferation and migration of vascular         smooth muscle cells can lead to neointimal thickening and         vascular restenosis. Excessive neointimal vascular smooth muscle         cell proliferation is associated with elevated expression of         IKCa1. Therefore, block of IKCa1 could represent a therapeutic         strategy to prevent restenosis after angioplasty.     -   ischaemia (KCa, Ca²⁺),—in neuronal or cardiac ischemia,         depolarization of cell membranes leads to opening of         voltage-gated sodium and calcium channels. In turn this can lead         to calcium overload, which is cytotoxic. Block of voltage-gated         sodium and/or calcium channels can reduce calcium overload and         provide cytoprotective effects. In addition, due to their         critical role in controlling and stabilizing cell membrane         potential, modulators of voltage- and calcium-activated         potassium channels can also act to reduce calcium overload and         protect cells.     -   renal incontinence (KCa), renal incontinence is associated with         overactive bladder smooth muscle cells. Calcium-activated         potassium channels are expressed in bladder smooth muscle cells,         where they control the membrane potential and indirectly control         the force and frequency of cell contraction. Openers of         calcium-activated potassium channels therefore provide a         mechanism to dampen electrical and contractile activity in         bladder, leading to reduced urge to urinate.     -   osteoporosis (Kv),     -   pain, including migraine (Na_(v), TRP [transient receptor         potential channels], P2X, Ca²⁺), N-type voltage-gated calcium         channels are key regulators of nociceptive neurotransmission in         the spinal cord. Ziconotide, a peptide blocker of N-type calcium         channels reduces nociceptive neurotransmission and is approved         worldwide for the symptomatic alleviation of severe chronic pain         in humans. Novel blockers of nociceptor-specific N-type calcium         channels would be improved analgesics with reduced side-effect         profiles.     -   hypertension (Ca²⁺),—L-type and T-type voltage-gated calcium         channels are expressed in vascular smooth muscle cells where         they control excitation-contraction coupling and cellular         proliferation. In particular, T-type calcium channel activity         has been linked to neointima formation during hypertension.         Blockers of L-type and T-type calcium channels are useful for         the clinical treatment of hypertension because they reduce         calcium influx and inhibit smooth muscle cell contraction.     -   wound healing, cell migration serves a key role in wound         healing. Intracellular calcium gradients have been implicated as         important regulators of cellular migration machinery in         keratinocytes and fibroblasts. In addition, ion flux across cell         membranes is associated with cell volume changes. By controlling         cell volume, ion channels contribute to the intracellular         environment that is required for operation of the cellular         migration machinery. In particular, IKCa1 appears to be required         universally for cell migration. In addition, Kv1.3, Kv3.1, NMDA         receptors and N-type calcium channels are associated with the         migration of lymphocytes and neurons.     -   stroke,     -   Alzheimer's,     -   Parkenson's Disease (nACHR, Nav)     -   Bipolar Disorder (Nav, Cav)     -   cancer, many potassium channel genes are amplified and protein         subunits are upregulated in many cancerous condition. Consistent         with a pathophysiological role for potassium channel         upregulation, potassium channel blockers have been shown to         suppress proliferation of uterine cancer cells and         hepatocarcinoma cells, presumably through inhibition of calcium         influx and effects on calcium-dependent gene expression.     -   a variety of neurological, cardiovascular, metabolic and         autoimmune diseases.

Both agonists and antagonists of ion channels can achieve therapeutic benefit. Therapeutic benefits can result, for example, from antagonizing Kv1.3, IKCa1, SKCa, BKCa, N-type or T-type Ca²⁺ channels and the like. Small molecule and peptide antagonists of these channels have been shown to possess utility in vitro and in vivo. Limitations in production efficiency and pharmacokinetics, however, have largely prevented clinical investigation of inhibitor peptides of ion channels.

Compositions of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv1.3, in particular OSK1 peptide analogs, whether or not conjugated to a half-life extending moiety, are useful as immunosuppressive agents with therapeutic value for autoimmune diseases. For example, such molecules are useful in treating multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, and rheumatoid arthritis. (See, e.g., H. Wulff et al. (2003) J. Clin. Invest. 111, 1703-1713 and H. Rus et al. (2005) PNAS 102, 11094-11099; Beeton et al., Targeting effector memory T cells with a selective inhibitor peptide of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005);1 Beeton et al. (2006), Kv1.3: therapeutic target for cell-mediated autoimmune disease, electronic preprint at //webfiles.uci.edu/xythoswfs/webui/2670029.1). Inhibitors of the voltage-gated potassium channel Kv1.3 have been examined in a variety of preclinical animal models of inflammation. Small molecule and peptide inhibitors of Kv1.3 have been shown to block delayed type hypersensitivity responses to ovalbumin [C. Beeton et al. (2005) Mol. Pharmacol. 67, 1369] and tetanus toxoid [G. C. Koo et al. (1999) Clin. Immunol. 197, 99]. In addition to suppressing inflammation in the skin, inhibitors also reduced antibody production [G. C. Koo et al. (1997) J. Immunol. 158, 5120]. Kv1.3 antagonists have shown efficacy in a rat adoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis (MS). The Kv1.3 channel is overexpressed on myelin-specific T cells from MS patients, lending further support to the utility Kv1.3 inhibitors may provide in treating MS. Inflammatory bone resorption was also suppressed by Kv1.3 inhibitors in a preclinical adoptive-transfer model of periodontal disease [P. Valverde et al. (2004) J. Bone Mineral Res. 19, 155]. In this study, inhibitors additionally blocked antibody production to a bacterial outer membrane protein,—one component of the bacteria used to induce gingival inflammation. Recently in preclinical rat models, efficacy of Kv1.3 inhibitors was shown in treating pristane-induced arthritis and diabetes [C. Beeton et al. (2006) preprint available at //webfiles.uci.edu/xythoswfs/webui/_xy-2670029_(—)1.]. The Kv1.3 channel is expressed on all subsets of T cells and B cells, but effector memory T cells and class-switched memory B cells are particularly dependent on Kv1.3 [H. Wulff et al. (2004) J. Immunol. 173, 776]. Gad5/insulin-specific T cells from patients with new onset type 1 diabetes, myelin-specific T cells from MS patients and T cells from the synovium of rheumatoid arthritis patients all overexpress Kv1.3 [C. Beeton et al. (2006) preprint at //webfiles.uci.edu/xythoswfs/webui/_xy-2670029_(—)1.]. Because mice deficient in Kv1.3 gained less weight when placed on a high fat diet [J. Xu et al. (2003) Human Mol. Genet. 12, 551] and showed altered glucose utilization [J. Xu et al. (2004) Proc. Natl. Acad. Sci. 101, 3112], Kv1.3 is also being investigated for the treatment of obesity and diabetes. Breast cancer specimens [M. Abdul et al. (2003) Anticancer Res. 23, 3347] and prostate cancer cell lines [S. P. Fraser et al. (2003) Pflugers Arch. 446, 559] have also been shown to express Kv1.3, and Kv1.3 blockade may be of utility for treatment of cancer. Disorders that can be treated in accordance with the inventive method of treating an autoimmune disorder, involving Kv1.3 inhibitor toxin peptide(s), include multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and systemic lupus erythematosus (SLE) and other forms of lupus.

Some of the cells that express the calcium-activated potassium of intermediate conductance IKCa1 include T cells, B cells, mast cells and red blood cells (RBCs). T cells and RBCs from mice deficient in IKCa1 show defects in volume regulation [T. Begenisich et al. (2004) J. Biol. Chem. 279, 47681]. Preclinical and clinical studies have demonstrated IKCa1 inhibitors utility in treating sickle cell anemia [J. W. Stocker et al. (2003) Blood 101, 2412]. Blockers of the IKCa1 channel have also been shown to block EAE, indicating they may possess utility in treatment of MS [E. P. Reich et al. (2005) Eur. J. Immunol. 35, 1027]. IgE-mediated histamine production from mast cells is also blocked by IKCa1 inhibitors [S. Mark Duffy et al. (2004) J. Allergy Clin. Immunol. 114, 66], therefore they may also be of benefit in treating asthma. The IKCa1 channel is overexpressed on activated T and B lymphocytes [H. Wulff et al. (2004) J. Immunol. 173, 776] and thus may show utility in treatment of a wide variety of immune disorders. Outside of the immune system, IKCa1 inhibitors have also shown efficacy in a rat model of vascular restinosis and thus might represent a new therapeutic strategy to prevent restenosis after angioplasty [R. Kohler et al. (2003) Circulation 108, 1119]. It is also thought that IKCa1 antagonists are of utility in treatment of tumor angiogenesis since inhibitors suppressed endothelial cell proliferation and angionenesis in vivo [I. Grgic et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25, 704]. The IKCa1 channel is upregulated in pancreatic tumors and inhibitors blocked proliferation of pancreatic tumor cell lines [H. Jager et al. (2004) Mol Pharmacol. 65, 630]. IKCa1 antagonists may also represent an approach to attenuate acute brain damage caused by traumatic brain injury [F. Mauler (2004) Eur. J. Neurosci. 20, 1761]. Disorders that can be treated with IKCa1 inhibitors include multiple sclerosis, asthma, psoriasis, contact-mediated dermatitis, rheumatoid & psoriatic arthritis, inflammatory bowel disease, transplant rejection, graft-versus-host disease, Lupus, restinosis, pancreatic cancer, tumor angiogenesis and traumatic brain injury.

Accordingly, molecules of this invention incorporating peptide antagonists of the calcium-activated potassium channel of intermediate conductance, IKCa can be used to treat immune dysfunction, multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus.

Accordingly, the present invention includes a method of treating an autoimmune disorder, which involves administering to a patient who has been diagnosed with an autoimmune disorder, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, a therapeutically effective amount of the inventive composition of matter, whereby at least one symptom of the disorder is alleviated in the patient. “Alleviated” means to be lessened, lightened, diminished, softened, mitigated (i.e., made more mild or gentle), quieted, assuaged, abated, relieved, nullified, or allayed, regardless of whether the symptom of interest is entirely erased, eradicated, eliminated, or prevented in a particular patient.

The present invention is further directed to a method of preventing or mitigating a relapse of a symptom of multiple sclerosis, which method involves administering to a patient, who has previously experienced at least one symptom of multiple sclerosis, a prophylactically effective amount of the inventive composition of matter, such that the at least one symptom of multiple sclerosis is prevented from recurring or is mitigated.

The inventive compositions of matter preferred for use in practicing the inventive method of treating an autoimmune disorder, e.g., inflammatory bowel disease (IBD, including Crohn's Disease and ulcerative colitis), and the method of preventing or mitigating a relapse of a symptom of multiple sclerosis include as P (conjugated as in Formula I), a Kv1.3 or IKCa1 antagonist peptide, such as a ShK peptide, an OSK1 peptide or an OSK1 peptide analog, a ChTx peptide and/or a Maurotoxin (MTx) peptide, or peptide analogs of any of these.

For example, the conjugated ShK peptide or ShK peptide analog can comprise an amino acid sequence selected from the following:

-   -   SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950,         or 1295 through 1300 as set forth in Table 2.

The conjugated OSK1 peptide, or conjugated or unconjugated OSK1 peptide analog, can comprise an amino acid sequence selected from the following:

-   -   SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through         1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID         NO: 1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK         (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7, or         any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006,         5009, 5010, and 5012 through 5015 as set forth in Table 7A,         Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G,         Table 7H, Table 7I, or Table 7J.

Also by way of example, a the conjugated MTX peptide, MTX peptide analog, ChTx peptide or ChTx peptide analog can comprise an amino acid sequence selected from:

-   -   SEQ ID NOS: 20, 330 through 343, 1301, 1302, 1304 through 1307,         1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13;         or SEQ ID NOS: 36, 59, 344 through 346, or 1369 through 1390 as         set forth in Table 14.

Also useful in these methods conjugated, or unconjugated, are a Kv1.3 or IKCa1 inhibitor toxin peptide analog that comprises an amino acid sequence selected from:

-   -   SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884         through 949, or 1295 through 1300 as set forth in Table 2; or     -   SEQ ID NOS: 980 through 1274,         GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO:         1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK         (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7; or     -   SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307,         1309, 1311, 1312, and 1315 through 1336 as set forth in Table         13.

In accordance with these inventive methods, a patient who has been diagnosed with an autoimmune disorder, such as, but not limited to multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoratic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, or a patient who has previously experienced at least one symptom of multiple sclerosis, are well-recognizable and/or diagnosed by the skilled practitioner, such as a physician, familiar with autoimmune disorders and their symptoms.

For example, symptoms of multiple sclerosis can include the following:

-   -   visual symptoms, such as, optic neuritis (blurred vision, eye         pain, loss of color vision, blindness); diplopia (double         vision); nystagmus (jerky eye movements); ocular dysmetria         (constant under- or overshooting eye movements); internuclear         opthalmoplegia (lack of coordination between the two eyes,         nystagmus, diplopia); movement and sound phosphenes (flashing         lights when moving eyes or in response to a sudden noise);         afferent pupillary defect (abnormal pupil responses);     -   motor symptoms, such as, paresis, monoparesis, paraparesis,         hemiparesis, quadraparesis (muscle weakness—partial or mild         paralysis); plegia, paraplegia, hemiplegia, tetraplegia,         quadraplegia (paralysis—total or near total loss of muscle         strength); spasticity (loss of muscle tone causing stiffness,         pain and restricting free movement of affected limbs);         dysarthria (slurred speech and related speech problems); muscle         atrophy (wasting of muscles due to lack of use); spasms, cramps         (involuntary contraction of muscles); hypotonia, clonus         (problems with posture); myoclonus, myokymia (jerking and         twitching muscles, tics); restless leg syndrome (involuntary leg         movements, especially bothersome at night); footdrop (foot drags         along floor during walking); dysfunctional reflexes (MSRs,         Babinski's, Hoffman's, Chaddock's);     -   sensory symptoms, such as, paraesthesia (partial numbness,         tingling, buzzing and vibration sensations); anaesthesia         (complete numbness/loss of sensation); neuralgia, neuropathic         and neurogenic pain (pain without apparent cause, burning,         itching and electrical shock sensations); L'Hermitte's (electric         shocks and buzzing sensations when moving head); proprioceptive         dysfunction (loss of awareness of location of body parts);         trigeminal neuralgia (facial pain);     -   coordination and balance symptoms, such as, ataxia (loss of         coordination); intention tremor (shaking when performing fine         movements); dysmetria (constant under- or overshooting limb         movements); vestibular ataxia (abnormal balance function in the         inner ear); vertigo (nausea/vomitting/sensitivity to travel         sickness from vestibular ataxia); speech ataxia (problems         coordinating speech, stuttering); dystonia (slow limb position         feedback); dysdiadochokinesia (loss of ability to produce         rapidly alternating movements, for example to move to a rhythm);     -   bowel, bladder and sexual symptoms, such as, frequent         micturation, bladder spasticity (urinary urgency and         incontinence); flaccid bladder, detrusor-sphincter dyssynergia         (urinary hesitancy and retention); erectile dysfunction (male         and female impotence); anorgasmy (inability to achieve orgasm);         retrograde ejaculation (ejaculating into the bladder); frigidity         (inability to become sexually aroused); constipation (infrequent         or irregular bowel movements); fecal urgency (bowel urgency);         fecal incontinence (bowel incontinence);     -   cognitive symptoms, such as, depression; cognitive dysfunction         (short-term and long-term memory problems, forgetfulness, slow         word recall); dementia; mood swings, emotional lability,         euphoria; bipolar syndrome; anxiety; aphasia, dysphasia         (impairments to speech comprehension and production); and     -   other symptoms, such as, fatigue; Uhthoff's Symptom (increase in         severity of symptoms with heat); gastroesophageal reflux (acid         reflux); impaired sense of taste and smell; epileptic seizures;         swallowing problems, respiratory problems; and sleeping         disorders.

By way of further example, symptoms of inflammatory bowel disease can include the following symptoms of Crohn's Disease or ulcerative colitis:

A. Symptoms of Crohn's disease can include:

-   -   Abdominal pain. The pain often is described as cramping and         intermittent, and the abdomen may be sore when touched.         Abdominal pain may turn to a dull, constant ache as the         condition progresses.     -   Diarrhea. Some patients may have diarrhea 10 to 20 times per         day. They may wake up at night and need to go to the bathroom.         Crohn's disease may cause blood in stools, but not always.     -   Loss of appetite.     -   Fever. In severe cases, fever or other symptoms that affect the         entire body may develop. A high fever may indicate a         complication involving infection, such as an abscess.     -   Weight loss. Ongoing symptoms, such as diarrhea, can lead to         weight loss. Too few red blood cells (anemia). Some patients         with Crohn's disease develop anemia because of low iron levels         caused by bloody stools or the intestinal inflammation itself.

B. The symptoms of ulcerative colitis may include:

-   -   Diarrhea or rectal urgency. Some patients may have diarrhea 10         to 20 times per day. The urge to defecate may wake patients at         night.     -   Rectal bleeding. Ulcerative colitis usually causes bloody         diarrhea and mucus. Patients also may have rectal pain and an         urgent need to empty the bowels.     -   Abdominal pain, often described as cramping. The patient's         abdomen may be sore when touched.     -   Constipation. This symptom may develop depending on what part of         the colon is affected.     -   Loss of appetite.     -   Fever. In severe cases, fever or other symptoms that affect the         entire body may develop.     -   Weight loss. Ongoing (chronic) symptoms, such as diarrhea, can         lead to weight loss.     -   Too few red blood cells (anemia). Some patients develop anemia         because of low iron levels caused by bloody stools or intestinal         inflammation.

The symptoms of multiple sclerosis and inflammatory bowel disease (including Crohn's Disease and ulcerative colitis) enumerated above, are merely illustrative and are not intended to be an exhaustive description of all possible symptoms experienced by a single patient or by several sufferers in composite, and to which the present invention is directed. Those skilled in the art are aware of various clinical symptoms and constellations of symptoms of autoimmune disorders suffered by individual patients, and to those symptoms are also directed the present inventive methods of treating an autoimmune disorder or of preventing or mitigating a relapse of a symptom of multiple sclerosis.

The therapeutically effective amount, prophylactically effective amount, and dosage regimen involved in the inventive methods of treating an autoimmune disorder or of preventing or mitigating a relapse of a symptom of multiple sclerosis, will be determined by the attending physician, considering various factors which modify the action of therapeutic agents, such as the age, condition, body weight, sex and diet of the patient, the severity of the condition being treated, time of administration, and other clinical factors. Generally, the daily amount or regimen should be in the range of about 1 to about 10,000 micrograms (μg) of the vehicle-conjugated peptide per kilogram (kg) of body mass, preferably about 1 to about 5000 μg per kilogram of body mass, and most preferably about 1 to about 1000 μg per kilogram of body mass.

Molecules of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv2.1 can be used to treat type II diabetes.

Molecules of this invention incorporating peptide antagonists of the M current (e.g., BeKm-1) can be used to treat Alzheimer's disease and enhance cognition.

Molecules of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv4.3 can be used to treat Alzheimer's disease.

Molecules of this invention incorporating peptide antagonists of the calcium-activated potassium channel of small conductance, SKCa can be used to treat epilepsy, memory, learning, neuropsychiatric, neurological, neuromuscular, and immunological disorders, schizophrenia, bipolar disorder, sleep apnea, neurodegeneration, and smooth muscle disorders.

Molecules of this invention incorporating N-type calcium channel antagonist peptides are useful in alleviating pain. Peptides with such activity (e.g., Ziconotide™, ω-conotoxin-MVIIA) have been clinically validated.

Molecules of this invention incorporating T-type calcium channel antagonist peptides are useful in alleviating pain. Several lines of evidence have converged to indicate that inhibition of Cav3.2 in dorsal root ganglia may bring relief from chronic pain. T-type calcium channels are found at extremely high levels in the cell bodies of a subset of neurons in the DRG; these are likely mechanoreceptors adapted to detect slowly-moving stimuli (Shin et al., Nature Neuroscience 6:724-730, 2003), and T-type channel activity is likely responsible for burst spiking (Nelson et al., J Neurosci 25:8766-8775, 2005). Inhibition of T-type channels by either mibefradil or ethosuximide reverses mechanical allodynia in animals induced by nerve injury (Dogrul et al., Pain 105:159-168, 2003) or by chemotherapy (Flatters and Bennett, Pain 109:150-161, 2004). Antisense to Cav3.2, but not Cav3.1 or Cav3.3, increases pain thresholds in animals and also reduces expression of Cav3.2 protein in the DRG (Bourinet et al., EMBO J 24:315-324, 2005). Similarly, locally injected reducing agents produce pain and increase Cav3.2 currents, oxidizing agents reduce pain and inhibit Cav3.2 currents, and peripherally administered neurosteroids are analgesic and inhibit T-type currents from DRG (Todorovic et al., Pain 109:328-339, 2004; Pathirathna et al., Pain 114:429-443, 2005). Accordingly, it is thought that inhibition of Cav3.2 in the cell bodies of DRG neurons can inhibit the repetitive spiking of these neurons associated with chronic pain states.

Molecules of this invention incorporating L-type calcium channel antagonist peptides are useful in treating hypertension. Small molecules with such activity (e.g., DHP) have been clinically validated.

Molecules of this invention incorporating peptide antagonists of the Na_(V)1 (TTX_(S)-type) channel can be used to alleviate pain. Local anesthetics and tricyclic antidepressants with such activity have been clinically validated. Such molecules of this invention can in particular be useful as muscle relaxants.

Molecules of this invention incorporating peptide antagonists of the Na_(V)1 (TTX_(R)-type) channel can be used to alleviate pain arising from nerve and or tissue injury.

Molecules of this invention incorporating peptide antagonists of glial & epithelial cell Ca²⁺-activated chloride channel can be used to treat cancer and diabetes.

Molecules of this invention incorporating peptide antagonists of NMDA receptors can be used to treat pain, epilepsy, brain and spinal cord injury.

Molecules of this invention incorporating peptide antagonists of nicotinic receptors can be used as muscle relaxants. Such molecules can be used to treat pain, gastric motility disorders, urinary incontinence, nicotine addiction, and mood disorders.

Molecules of this invention incorporating peptide antagonists of 5HT3 receptor can be used to treat Nausea, pain, and anxiety.

Molecules of this invention incorporating peptide antagonists of the norepinephrine transporter can be used to treat pain, anti-depressant, learning, memory, and urinary incontinence.

Molecules of this invention incorporating peptide antagonists of the Neurotensin receptor can be used to treat pain.

In addition to therapeutic uses, the compounds of the present invention can be useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest (e.g., a receptor) that is capable of being activated comprising the steps of: (a) contacting the sample with a compound of this invention; and (b) detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention can be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow for detection. The compounds are useful for identifying normal or abnormal proteins of interest.

The therapeutic methods, compositions and compounds of the present invention can also be employed, alone or in combination with other molecules in the treatment of disease.

Pharmaceutical Compositions

In General. The present invention also provides pharmaceutical compositions comprising the inventive composition of matter and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be configured for administration to a patient by a wide variety of delivery routes, e.g., an intravascular delivery route such as by injection or infusion, subcutaneous, intramuscular, intraperitoneal, epidural, or intrathecal delivery routes, or for oral, enteral, pulmonary (e.g., inhalant), intranasal, transmucosal (e.g., sublingual administration), transdermal or other delivery routes and/or forms of administration known in the art. The inventive pharmaceutical compositions may be prepared in liquid form, or may be in dried powder form, such as lyophilized form. For oral or enteral use, the pharmaceutical compositions can be configured, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs or enteral formulas.

In the practice of this invention the “pharmaceutically acceptable carrier” is any physiologically tolerated substance known to those of ordinary skill in the art useful in formulating pharmaceutical compositions, including, any pharmaceutically acceptable diluents, excipients, dispersants, binders, fillers, glidants, anti-frictional agents, compression aids, tablet-disintegrating agents (disintegrants), suspending agents, lubricants, flavorants, odorants, sweeteners, permeation or penetration enhancers, preservatives, surfactants, solubilizers, emulsifiers, thickeners, adjuvants, dyes, coatings, encapsulating material(s), and/or other additives singly or in combination. Such pharmaceutical compositions can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are herein incorporated by reference in their entirety. The compositions can be prepared in liquid form, or can be in dried powder, such as lyophilized form. Implantable sustained release formulations are also useful, as are transdermal or transmucosal formulations. Additionally (or alternatively), the present invention provides compositions for use in any of the various slow or sustained release formulations or microparticle formulations known to the skilled artisan, for example, sustained release microparticle formulations, which can be administered via pulmonary, intranasal, or subcutaneous delivery routes.

One can dilute the inventive compositions or increase the volume of the pharmaceutical compositions of the invention with an inert material. Such diluents can include carbohydrates, especially, mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

A variety of conventional thickeners are useful in creams, ointments, suppository and gel configurations of the pharmaceutical composition, such as, but not limited to, alginate, xanthan gum, or petrolatum, may also be employed in such configurations of the pharmaceutical composition of the present invention. A permeation or penetration enhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide, N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or 3-hydroxy-N-methyl-2-pyrrolidone can also be employed. Useful techniques for producing hydrogel matrices are known. (E.g., Feijen, Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents, U.S. Pat. No. 4,925,677; Shah et al., Biodegradable pH/thermosensitive hydrogels for sustained delivery of biologically active agents, WO 00/38651 A1). Such biodegradable gel matrices can be formed, for example, by crosslinking a proteinaceous component and a polysaccharide or mucopolysaccharide component, then loading with the inventive composition of matter to be delivered.

Liquid pharmaceutical compositions of the present invention that are sterile solutions or suspensions can be administered to a patient by injection, for example, intramuscularly, intrathecally, epidurally, intravascularly (e.g., intravenously or intraarterially), intraperitoneally or subcutaneously. (See, e.g., Goldenberg et al., Suspensions for the sustained release of proteins, U.S. Pat. No. 6,245,740 and WO 00/38652 A1). Sterile solutions can also be administered by intravenous infusion. The inventive composition can be included in a sterile solid pharmaceutical composition, such as a lyophilized powder, which can be dissolved or suspended at a convenient time before administration to a patient using sterile water, saline, buffered saline or other appropriate sterile injectable medium.

Implantable sustained release formulations are also useful embodiments of the inventive pharmaceutical compositions. For example, the pharmaceutically acceptable carrier, being a biodegradable matrix implanted within the body or under the skin of a human or non-human vertebrate, can be a hydrogel similar to those described above. Alternatively, it may be formed from a poly-alpha-amino acid component. (Sidman, Biodegradable, implantable drug delivery device, and process for preparing and using same, U.S. Pat. No. 4,351,337). Other techniques for making implants for delivery of drugs are also known and useful in accordance with the present invention.

In powder forms, the pharmaceutically acceptable carrier is a finely divided solid, which is in admixture with finely divided active ingredient(s), including the inventive composition. For example, in some embodiments, a powder form is useful when the pharmaceutical composition is configured as an inhalant. (See, e.g., Zeng et al., Method of preparing dry powder inhalation compositions, WO 2004/017918; Trunk et al., Salts of the CGRP antagonist BIBN4096 and inhalable powdered medicaments containing them, U.S. Pat. No. 6,900,317).

One can dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts can also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo™, Emdex™, STA-Rx™ 1500, Emcompress™ and Avicell™.

Disintegrants can be included in the formulation of the pharmaceutical composition into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab™. Sodium starch glycolate, Amberlite™, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite can all be used. Insoluble cationic exchange resin is another form of disintegrant. Powdered gums can be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders can be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent can be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants can be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants can also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants can include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Oral dosage forms. Also useful are oral dosage forms of the inventive compositions. If necessary, the composition can be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached half-life extending moieties in this invention can also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis (1981), Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (Hocenberg and Roberts, eds.), Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods.”

In one embodiment, the pharmaceutically acceptable carrier can be a liquid and the pharmaceutical composition is prepared in the form of a solution, suspension, emulsion, syrup, elixir or pressurized composition. The active ingredient(s) (e.g., the inventive composition of matter) can be dissolved, diluted or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as detergents and/or solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers, buffers at appropriate pH (e.g., Tris-HCl, acetate, phosphate), adjuvants, anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), sweeteners, flavoring agents, suspending agents, thickening agents, bulking substances (e.g., lactose, mannitol), colors, viscosity regulators, stabilizers, electrolytes, osmolutes or osmo-regulators. Additives can also be included in the formulation to enhance uptake of the inventive composition. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

Useful are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences (1990), supra, in Chapter 89, which is hereby incorporated by reference in its entirety. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation can be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation can be used and the liposomes can be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, in Chapter 10, which is hereby incorporated by reference in its entirety. In general, the formulation will include the inventive compound, and inert ingredients that allow for protection against the stomach environment, and release of the biologically active material in the intestine.

The composition of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents can all be included. For example, the protein (or derivative) can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

In tablet form, the active ingredient(s) are mixed with a pharmaceutically acceptable carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain up to 99% of the active ingredient(s). Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Controlled release formulation can be desirable. The composition of this invention could be incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices can also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compositions of this invention is by a method based on the Oros™ therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

Other coatings can be used for the formulation. These include a variety of sugars that could be applied in a coating pan. The therapeutic agent could also be given in a film-coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating. Film coating can be carried out in a pan coater or in a fluidized bed or by compression coating.

Pulmonarv delivery forms. Pulmonary delivery of the inventive compositions is also useful. The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins,” Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Useful in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass. (See, e.g., Helgesson et al., Inhalation device, U.S. Pat. No. 6,892,728; McDerment et al., Dry powder inhaler, WO 02/11801 A1; Ohki et al., Inhalant medicator, U.S. Pat. No. 6,273,086).

All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.

Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations can include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants can be used. PEG can be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, can be used. Bile salts and other related enhancers can be used. Cellulose and cellulose derivatives can be used. Amino acids can be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation can also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid can also be useful as a surfactant. (See, e.g., Bäckström et al., Aerosol drug formulations containing hydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. 6,932,962).

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and can also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery forms. In accordance with the present invention, intranasal delivery of the inventive composition of matter and/or pharmaceutical compositions is also useful, which allows passage thereof to the blood stream directly after administration to the inside of the nose, without the necessity for deposition of the product in the lung. Formulations suitable for intransal administration include those with dextran or cyclodextran, and intranasal delivery devices are known. (See, e.g, Freezer, Inhaler, U.S. Pat. No. 4,083,368).

Transdermal and transmucosal (e.g., buccal) delivery forms). In some embodiments, the inventive composition is configured as a part of a pharmaceutically acceptable transdermal or transmucosal patch or a troche. Transdermal patch drug delivery systems, for example, matrix type transdermal patches, are known and useful for practicing some embodiments of the present pharmaceutical compositions. (E.g., Chien et al., Transdermal estrogen/progestin dosage unit, system and process, U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary et al., Diffusion matrix for transdermal drug administration and transdermal drug delivery devices including same, U.S. Pat. No. 4,911,916; Teillaud et al., EVA-based transdermal matrix system for the administration of an estrogen and/or a progestogen, U.S. Pat. No. 5,605,702; Venkateshwaran et al., Transdermal drug delivery matrix for coadministering estradiol and another steroid, U.S. Pat. No. 5,783,208; Ebert et al., Methods for providing testosterone and optionally estrogen replacement therapy to women, U.S. Pat. No. 5,460,820). A variety of pharmaceutically acceptable systems for transmucosal delivery of therapeutic agents are also known in the art and are compatible with the practice of the present invention. (E.g., Heiber et al., Transmucosal delivery of macromolecular drugs, U.S. Pat. Nos. 5,346,701 and 5,516,523; Longenecker et al., Transmembrane formulations for drug administration, U.S. Pat. No. 4,994,439).

Buccal delivery of the inventive compositions is also useful. Buccal delivery formulations are known in the art for use with peptides. For example, known tablet or patch systems configured for drug delivery through the oral mucosa (e.g., sublingual mucosa), include some embodiments that comprise an inner layer containing the drug, a permeation enhancer, such as a bile salt or fusidate, and a hydrophilic polymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch, gelatin, or any number of other polymers known to be useful for this purpose. This inner layer can have one surface adapted to contact and adhere to the moist mucosal tissue of the oral cavity and can have an opposing surface adhering to an overlying non-adhesive inert layer. Optionally, such a transmucosal delivery system can be in the form of a bilayer tablet, in which the inner layer also contains additional binding agents, flavoring agents, or fillers. Some useful systems employ a non-ionic detergent along with a permeation enhancer. Transmucosal delivery devices may be in free form, such as a cream, gel, or ointment, or may comprise a determinate form such as a tablet, patch or troche. For example, delivery of the inventive composition can be via a transmucosal delivery system comprising a laminated composite of, for example, an adhesive layer, a backing layer, a permeable membrane defining a reservoir containing the inventive composition, a peel seal disc underlying the membrane, one or more heat seals, and a removable release liner. (E.g., Ebert et al., Transdermal delivery system with adhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang et al., Device for administering an active agent to the skin or mucosa, U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are merely illustrative of available transmucosal drug delivery technology and are not limiting of the present invention.

Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.

WORKING EXAMPLES

The compositions described above can be prepared as described below. These examples are not to be construed in any way as limiting the scope of the present invention.

Example 1 Fc-L10-ShK[1-35] Mammalian Expression

Fc-L10-ShK[1-35], also referred to as “Fc-2xL-ShK[1-35]”, an inhibitor of Kv1.3. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a linker sequence and a monomer of the Kv1.3 inhibitor peptide ShK[1-35] was constructed as described below. Methods for expressing and purifying the peptibody from mammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosed herein.

The expression vector pcDNA3.1(+) CMVi (FIG. 13A) was constructed by replacing the CMV promoter between MluI and HindIII in pcDNA3.1(+) with the CMV promoter plus intron (Invitrogen). The expression vector pcDNA3.1 (+) CMVi-hFc-ActivinRIIB (FIG. 13B) was generated by cloning a HindIII-NotI digested PCR product containing a 5′ Kozak sequence, a signal peptide and the human Fc-linker-ActivinRIIB fusion protein with the large fragment of HindIII-NotI digested pcDNA3.1(+) CMVi. The nucleotide and amino acid sequence of the human IgG1 Fc region in pcDNA3.1(+) CMVi-hFc-ActivinRIIB is shown in FIG. 3A-3B. This vector also has a GGGGSGGGGS (“L10”; SEQ ID NO:79) linker split by a BamHI site thus enabling with the oligo below formation of the 10 amino acid linker between Fc and the ShK[1-35] peptide (see FIG. 14A-14B) for the final Fc-L10-ShK[1-35] nucleotide and amino acid sequence (FIG. 14A-14B and SEQ ID NO: 77 and SEQ ID NO:78).

The Fc-L10-ShK[1-35] expression vector was constructing using PCR stategies to generate the full length ShK gene linked to a four glycine and one serine amino acid linker (lower case letters here indicate linker sequence of L-form amino acid residues) with two stop codons and flanked by BamHI and NotI restriction sites as shown below.

BamHI GGATCCGGAGGAGGAGGAAGCCGCAGC SEQ ID NO: 657       g  g  g  g  s  R  S SEQ ID NO: 658 TGCATCGACACCATCCCCAAGAGCCGC C  I  D  T  I  P  K  S  R TGCACCGCCTTCCAG C  T  A  F  Q TGCAAGCACAGCATGAAGTACCGCCTG C  K  H  S  M  K  Y  R  L AGCTTCTGCCGCAAGACCTGCGGCACC S  F  C  R  K  T  C  G  T TGCTAATGAGCGGCCGC// C        NotI//

Two oligos with the sequence as depicted below were used in a PCR reaction with Herculase™ polymerase (Stratagene) at 94° C.-30 sec, 50° C.-30 sec, and 72° C.-1 min for 30 cycles.

cat gga tcc gga gga gga gga SEQ ID NO: 659 agc cgc agc tgc atc gac acc atc ccc aag agc cgc tgc acc gcc ttc cag tgc aag cac// cat gcg gcc gct cat tag cag gtg SEQ ID NO: 660 ccg cag gtc ttg cgg cag aag ctc agg cgg tac ttc atg ctg tgc ttg cac tgg aag g//

The resulting PCR products were resolved as the 150 bp bands on a one percent agarose gel. The 150 bp PCR product was digested with BamHI and NotI (Roche) restriction enzymes and agarose gel purified by Gel Purification Kit (Qiagen). At the same time, the pcDNA3.1 (+) CMVi-hFc-ActivinRIIB vector (FIG. 13B) was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Purification Kit. The gel purified PCR fragment was ligated to the purified large fragment and transformed into XL-1 blue bacteria (Stratagene). DNAs from transformed bacterial colonies were isolated and digested with BamHI and NotI restriction enzyme digestion and resolved on a one percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification. The DNA from Fc-2xL-ShK in pcDNA3.1(+) CMVi clone was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the predicted coding sequence, which is shown in FIG. 14A-14B.

HEK-293 cells used in transient transfection expression of Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi protein were cultured in growth medium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS from Gibco) and 1× non-essential amino acid (NEAA from Gibco). 5.6 ug of Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi plasmid that had been phenol/chloroform extracted was transfected into HEK-293 cells using Fugene 6 (Roche). The cells recovered for 24 hours, and then placed in DMEM High Glucose and 1×NEAA medium for 48 hours. The conditioned medium was concentrated 50× by running 30 ml through Centriprep YM-10 filter (Amicon) and further concentrated by a Centricon YM-10 (Amicon) filter. Various amounts of concentrated medium were mixed with an in-house 4× Loading Buffer (without B-mercaptoethanol) and electrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2 hours in a 5× Tank buffer solution (0.123 Tris Base, 0.96M Glycine) along with 10 ul of BenchMark Pre-Stained Protein ladder (Invitrogen). The gel was then soaked in Electroblot buffer (35 mM Tris base, 20% methanol, 192 mM glycine) for 30 minutes. A PVDF membrane from Novex (Cat. No. LC2002, 0.2 um pores size) was soaked in methanol for 30 seconds to activate the PVDF, rinsed with deionized water, and soaked in Electroblot buffer. The pre-soaked gel was blotted to the PVDF membrane using the XCell II Blot module according to the manufacturer instructions (Novex) at 40 mA for 2 hours. Then, the blot was first soaked in a 5% milk (Carnation) in Tris buffered saline solution pH7.5 (TBS) for 1 hour at room temperature and incubated with 1:500 dilution in TBS with 0.1% Tween-20 (TBST Sigma) and 1% milk buffer of the HRP-conjugated murine anti-human Fc antibody (Zymed Laboratores Cat. no. 05-3320) for two hours shaking at room temperature. The blot was then washed three times in TBST for 15 minutes per wash at room temperature. The primary antibody was detected using Amersham Pharmacia Biotech's ECL western blotting detection reagents according to manufacturer's instructions. Upon ECL detection, the western blot analysis displayed the expected size of 66 kDa under non-reducing gel conditions (FIG. 24A).

AM1 CHOd- (Amgen Proprietary) cells used in the stable expression of Fc-L10-ShK[1-35] protein were cultured in AM1 CHOd- growth medium containing DMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HT from Gibco) and 1×NEAA. 6.5 ug of pcDNA3.1(+) CMVi-Fc-ShK plasmid was also transfected into AM1 CHOd- cells using Fugene 6. The following day, the transfected cells were plated into twenty 15 cm dishes and selected using DMEM high glucose, 10% FBS, 1×HT, 1×NEAA and Geneticin (800 μg/ml G418 from Gibco) for thirteen days. Forty-eight surviving colonies were picked into two 24-well plates. The plates were allowed to grow up for a week and then replicated for freezing. One set of each plate was transferred to AM1 CHOd- growth medium without 10% FBS for 48 hours and the conditioned media were harvested. Western Blot analysis similar to the transient Western blot analysis with detection by the same anti-human Fc antibody was used to screen 15 ul of conditioned medium for expressing stable CHO clones. Of the 48 stable clones, more than 50% gave ShK expression at the expected size of 66 kDa. The BB6, BD5 and BD6 clones were selected with BD5 and BD6 as a backup to the primary clone BB6 (FIG. 24B).

The BB6 clone was scaled up into ten roller bottles (Corning) using AM1 CHOd- growth medium and grown to confluency as judged under the microscope. Then, the medium was exchanged with a serum-free medium containing to 50% DMEM high glucose and 50% Ham's F12 (Gibco) with 1×HT and 1×NEAA and let incubate for one week. The conditioned medium was harvested at the one-week incubation time, filtered through 0.45 μm filter (Corning) and frozen. Fresh serum-free medium was added and incubated for an additional week. The conditioned serum-free medium was harvested like the first time and frozen.

Purification of monovalent and bivalent dimeric Fc-L10-ShK(1-35). Approximately 4 L of conditioned medium was thawed in a water bath at room temperature. The medium was concentrated to about 450 ml using a Satorius Sartocon Polysulfon 10 tangential flow ultra-filtration cassette (0.1 m²) at room temperature. The retentate was then filtered through a 0.22 μm cellulose acetate filter with a pre-filter. The retentate was then loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9 ml) was diluted to 50 ml with water and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 5 ml/min and 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 25% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 3.4 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device and then filtered though a Costar 0.22 μm cellulose acetate syringe filter.

A spectral scan was then conducted on 10 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 26A). The concentration of the filtered material was determined to be 5.4 mg/ml using a calculated molecular mass of 32,420 g/mol and extinction coefficient of 47,900 M⁻¹cm⁻¹.

The purity of the filtered bivalent dimeric Fc-L10-ShK(1-35) product was assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 26B). The monvalent dimeric Fc-L10-ShK(1-35) product was analyzed using reducing and non-reducing sample buffers by SDS-PAGE on a 1.0 mm TRIS-glycine 4-20% gel developed at 220 V and stained with Boston Biologicals QuickBlue (FIG. 26E). The endotoxin levels were then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 108-fold dilution of the sample in PBS yielding a result of <1 EU/mg protein. The macromolecular state of the products was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 26C, bivalent dimeric Fc-L10-ShK(1-35); FIG. 26F, monovalent dimeric Fc-L10-SHK(1-35)). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 26D) and confirmed (within experimental error) the integrity of the purified peptibody. The product was then stored at −80° C.

Purified bivalent dimeric Fc-L10-ShK[1-35] potently blocked human Kv1.3 (FIG. 30A and FIG. 30B) as determined by electrophysiology (see Example 36). The purified bivalent dimeric Fc-L10-ShK[1-35] molecule also blocked T cell proliferation (FIG. 36A and FIG. 36B) and production of the cytokines IL-2 (FIG. 35A and FIG. 37A) and IFN-g (FIG. 35B and FIG. 37B).

Example 2 Fc-L-ShK[2-35] Mammalian Expression

A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the Kv1.3 inhibitor peptide ShK[2-35] was constructed using standard PCR technology. The ShK[2-35] and the 5, 10, or 25 amino acid linker portion of the molecule were generated in a PCR reaction using the original Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi as a template (Example 1, FIG. 14A-14B). All ShK constructs should have the following amino acid sequence of SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC  (SEQ ID NO: 92) with the first amino acid of the wild-type sequence deleted.

The sequences of the primers used to generate Fc-L5-ShK[2-35], also referred to as “Fc-1×L-ShK[2-35]”, are shown below:

cat gga tcc agc tgc atc gac SEQ ID NO: 661 acc atc//; cat gcg gcc gct cat tag c//; SEQ ID NO: 662

The sequences of the primers used to generate Fc-L10-ShK[2-35], also referred to as “Fc-2×L-ShK[2-35]” are shown below:

cat gga tcc gga gga gga gga SEQ ID NO: 663 agc agc tgc a//; cat gcg gcc gct cat tag cag SEQ ID NO: 664 gtg c//;

The sequences of the primers used to generate Fc-L25-ShK[2-35], also referred to as “Fc-5×L-ShK[2-35]”, are shown below:

cat gga tcc ggg ggt ggg ggt SEQ ID NO: 665 tct ggg ggt ggg ggt tct gga gga gga gga agc gga gga gga gga agc agc tgc a//; cat gcg gcc gct cat tag cag SEQ ID NO: 666 gtg c//;

The PCR products were digested with BamHI and NotI (Roche) restriction enzymes and agarose gel purified by Gel Purification Kit. At the same time, the pcDNA3.1(+) CMVi-hFc-ActivinRIIB vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Purification Kit. Each purified PCR product was ligated to the large fragment and transformed into XL-1 blue bacteria. DNAs from transformed bacterial colonies were isolated and subjected to BamHI and NotI restriction enzyme digestions and resolved on a one percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification. The DNA from this clone was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the expected sequence.

Plasmids containing the Fc-1xL-Shk[2-35], Fc-2xL-Shk[2-35] and Fc-5xL-Shk[2-35] inserts in pcDNA3.1 (+) CMVi vector were digested with Xba1 and Xho1 (Roche) restriction enzymes and gel purified. The inserts were individually ligated into Not1 and SalI (Roche) digested pDSRα-22 (Amgen Proprietary) expression vector. Integrity of the resulting constructs were confirmed by DNA sequencing. The final plasmid DNA expression vector constructs were pDSRα-22-Fc-1xL-Shk[2-35], pDSRα-22-Fc-2xL-Shk[2-35] (FIG. 13C and FIG. 15A-15B) and pDSRα-22-Fc-5xL-Shk[2-35] (FIG. 16A-16B) and contained 5, 10 and 25 amino acid linkers, respectively.

Twenty-four hours prior to transfection, 1.2e7 AM-1/D CHOd- (Amgen Proprietary) cells were plated into a T-175 cm sterile tissue culture flask, to allow 70-80% confluency on the day of transfection. The cells had been maintained in the AM-1/D CHOd- culture medium containing DMEM High Glucose, 5% FBS, 1× Glutamine Pen/Strep (Gibco), 1×HT, 1×NEAA's and 1× sodium pyruvate (Gibco). The following day, eighteen micrograms of each of the linearized pDSRα22:Fc-1xL-ShK[2-35], pDSRα22:Fc-2xL-ShK[2-35] and pDSRα22:Fc-5xL-ShK[2-35] (RDS's #20050037685, 20050053709, 20050073295) plasmids were mixed with 72 μg of linearized Selexis MAR plasmid and pPAGO1 (RDS 20042009896) and diluted into 6 ml of OptiMEM in a 50 ml conical tube and incubate for five minutes. LF2000 (210 μl) was added to 6 ml of OptiMEM and incubated for five minutes. The diluted DNA and LF2000 were mixed together and incubated for 20 minutes at room temperature. In the meantime, the cells were washed one time with PBS and then 30 ml OptiMEM without antibiotics were added to the cells. The OptiMEM was aspirated off, and the cells were incubated with 12 ml of DNA/LF2000 mixture for 6 hours or overnight in the 37° C. incubator with shaking. Twenty-four hours post transfection, the cells were split 1:5 into AM-1/D CHOd- culture medium and at differing dilutions for colony selection. Seventy-two hours post transfection, the cell medium was replaced with DHFR selection medium containing 10% Dialyzed FBS (Gibco) in DMEM High Glucose, plus 1× Glutamine Pen/Strep, 1×NEAA's and 1× Na Pyr to allow expression and secretion of protein into the cell medium. The selection medium was changed two times a week until the colonies are big enough to pick. The pDSRa22 expression vector contains a DHFR expression cassette, which allows transfected cells to grow in the absence of hypoxanthine and thymidine. The five T-175 pools of the resulting colonies were scaled up into roller bottles and cultured under serum free conditions. The conditioned media were harvested and replaced at one-week intervals. The resulting 3 liters of conditioned medium was filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.) and transferred to Protein Chemistry for purification. As a backup, twelve colonies were selected from the 10 cm plates after 10-14 days on DHFR selection medium and expression levels evaluated by western blot using HRP conjugated anti human IgGFc as a probe. The three best clones expressing the highest level of each of the different linker length Fc-L-ShK[2-35] fusion proteins were expanded and frozen for future use.

Purification of Fc-L10-ShK(2-35). Approximately 1 L of conditioned medium was thawed in a water bath at room temperature. The medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 8.5 ml) combined with 71 μl 3 M sodium acetate and then diluted to 50 ml with water. The diluted material was then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then filtered through a 0.22 μm cellulose acetate filter and concentrated to about 3.9 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. A spectral scan was then conducted on 10 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27E). The concentration of the filtered material was determined to be 2.76 mg/ml using a calculated molecular mass of 30,008 g/mol and extinction coefficient of 36,900 M⁻¹cm⁻¹. Since material was found in the permeate, repeated concentration step on the permeate using a new Macrosep cartridge. The new batch of concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. Both lots of concentrated material were combined into one pool.

A spectral scan was then conducted on 10 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer. The concentration of the filtered material was determined to be 3.33 mg/ml using a calculated molecular mass of 30,008 g/mol and extinction coefficient of 36,900 M⁻¹cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample in PBS yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27B). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27F) and the experiment confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.

FIG. 31B shows that purified Fc-L10-ShK[2-35] potently blocks human Kv1.3 current (electrophysiology was done as described in Example 36). The purified Fc-L10-ShK[2-35] molecule also blocked IL-2 (FIG. 64A and FIG. 64B) and IFN-g (FIG. 65A and FIG. 65B) production in human whole blood, as well as, upregulation of CD40L (FIG. 66A and FIG. 66B) and IL-2R (FIG. 67A and FIG. 67B) on T cells.

Purification of Fc-L5-ShK(2-35). Approximately 1 L of conditioned medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9 ml) combined with 450 μl 1 M tris HCl pH 8.5 followed by 230 μl 2 M acetic acid and then diluted to 50 ml with water. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 5.5 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature.

A spectral scan was then conducted on 10 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27G). The concentration of the filtered material was determined to be 4.59 mg/ml using a calculated molecular mass of 29,750 g/mol and extinction coefficient of 36,900 M⁻¹cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27C). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 92-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27H). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27I) and confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.

FIG. 31C shows that purified Fc-L5-ShK[2-35] is highly active and blocks human Kv1.3 as determined by whole cell patch clamp electrophysiology (see Example 36).

Purification of Fc-L25-ShK(2-35). Approximately 1 L of conditioned medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9.5 ml) combined with 119 μl 3 M sodium acetate and then diluted to 50 ml with water. The pH adjusted material was then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions containing the main peak from the chromatogram were pooled and filtered through a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer FIG. 27J. The concentration of the filtered material was determined to be 1.40 mg/ml using a calculated molecular mass of 31,011 g/mol and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27D). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 28-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27K). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27L) and this confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.

Purified Fc-L25-ShK[2-35] inhibited human Kv1.3 with an IC₅₀ of ˜150 pM by whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells (Example 36).

Example 3 Fc-L-ShK[1-35] Bacterial Expression

Description of bacterial peptibody expression vectors and procedures for cloning and expression of peptibodies. The cloning vector used for bacterial expression (Examples 3-30) is based on pAMG21 (originally described in U.S. Patent 2004/0044188). It has been modified in that the kanamycin resistance component has been replaced with ampicillin resistance by excising the DNA between the unique BstBI and NsiI sites of the vector and replacing with an appropriately digested PCR fragment bearing the beta-lactamase gene using PCR primers CCA ACA CAC TTC GAA AGA CGT TGA TCG GCA C (SEQ ID NO: 667) and CAC CCA ACA ATG CAT CCT TAA AAA AAT TAC GCC C (SEQ ID NO: 668) with pUC19 DNA as the template source of the beta-lactamase gene conferring resistance to ampicillin. The new version is called pAMG21ampR.

Description of cloning vector pAMG21ampR-Fc-Pep used in examples 3 to 30, excluding 15 and 16. FIG. 11A-C and FIG. 11D (schematic diagram) show the ds-DNA that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the C-terminus of the Fc gene. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. This entire region of DNA is shown in FIG. 11A-C. The coding region for Fc extends from nt 5134 to 5817 and the protein sequence appears below the DNA sequence. This is followed in frame by a glyX5 linker (nt's 5818-5832). A BsmBI site (GAGACG) spansnucleotides 5834-5839. DNA cleavage occurs between nucleotides 5828 and 5829 on the upper DNA strand and between nucleotides 5832 and 5833 on the lower DNA strand. Digestion creates 4 bp cohesive termini as shown here. The BsmBI site is underlined.

AGGTGG TGGTTGAGACG SEQ ID NO: 683 TCCACCACCA     ACTCTGC SEQ ID NO: 684

A second BsmBI site occurs at nucleotides 6643 through 6648; viz., CGTCTC. DNA cleavage occurs between nucleotides 6650 and 6651 on the upper strand and between 6654 and 6655 on the lower strand.

CGTCTCT TAAGGATCCG SEQ ID NO: 685 GCAGAGAATTC     CTAGGC SEQ ID NO: 686

Between the two BsmBI sites is a dispensable chloramphenicol resistance cassette constitutively expressing chloramphenicol acetyltransferase (cat gene). The cat protein sequence:

1 MEKKITGYTT VDISQWHRKE HFEAFQS SEQ ID NO: 1337 VAQCTYNQTVQLD ITAFLKTVKK 51 NKHKFYPAFI HILARLMNAH PEFRMAM KDGELVIWDSVHP CYTVFHEQTE 101 TFSSLWSEYH DDFRQFLHIY SQDVACY GENLAYFPKGFIE NMFFVSANPW 151 VSFTSFDLNV ANMDNFFAPV FTMGKYY TQGDKVLMPLAIQ VHHAVCDGFH 201 VGRMLNELQQ YCDEWQGGA// is shown in FIG. 11A-C and extends from nucleotides 5954 to 6610. The peptide encoding duplexes in each example (except Examples 15 and 16) bear cohesive ends complementary to those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in examples 15 and 16. FIG. 12A-C, and the schematic diagram in FIG. 12D, shows the ds-DNA sequence that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the N-terminus of the Fc gene. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. The coding region for Fc extends from nt 5640 to 6309 and the protein sequence appears below the DNA sequence. This is preceded in frame by a glyX5 linker (nt's 5614-5628). A BsmBI site spans nucleotides 5138 to 5143; viz., GAGACG. The cutting occurs between nucleotides 5132 and 5133 on the upper DNA strand and between 5136 and 5137 on the lower DNA strand.

Digestion creates 4 bp cohesive termini as shown. The BsmBI site is underlined.

AATAACA TATGCGAGACG SEQ ID NO: 687 TTATTGTATAC     GCTCTGC SEQ ID NO: 688

A second BsmBI site occurs at nucleotides 5607 through 5612; viz., CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upper strand and between 5617 and 5618 on the lower strand.

CGTCTCA GGTGGTGGT GCAGAGTCCAC     CACCA SEQ ID NO: 689

Between the BsmBI sites is a dispensable zeocin resistance cassette constitutively expressing the Shigella ble protein. The ble protein sequence:

1 MAKLTSAVPV LTARDVAGAV SEQ ID NO: 1338 EFWTDRLGFS RDFVEDDFAG VVRDDVTLFI 51 SAVQDQVVPD NTLAWVWVRG LDELYAEWSE VVSTNFRDAS GPAMTEIGEQ 101 PWGREFALRD PAGNCVHFVA EEQD// is shown extending from nucleotides 5217 to 5588 in FIG. 12A-C. The peptide encoding duplexes in Examples 15 and 16 bear cohesive ends complementary to those presented by the vector.

Description of the cloning vector DAMG21ampR-Pep-Fc used in Examples 52 and 53. FIG. 12E-G shows the ds-DNA sequence that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the N-terminus of the Fc gene in which the first two codons of the peptide are to be met-gly. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. The coding region for Fc extends from nt 5632 to 6312 and the protein sequence appears below the DNA sequence. This is preceded in frame by a glyX5 linker (nt's 5617-5631). A BsmBI site spans nucleotides 5141 to 5146; viz., GAGACG. The cutting occurs between nucleotides 5135 and 5136 on the upper DNA strand and between 5139 and 5140 on the lower DNA strand.

Digestion creates 4 bp cohesive termini as shown. The BsmBI site is underlined.

AATAACATAT GGGTCGAGACG SEQ ID NO: 1344 SEQ ID NO: 1343 TTATTGTATACCCA     GCTCTGC SEQ ID NO: 1345 A second BsmBI site occurs at nucleotides 5607 through 5612; viz., CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upper strand and between 5617 and 5618 on the lower strand.

CGTCTCA GGTGGTGGT GCAGAGTCCAC     CACCA SEQ ID NO: 1346 Between the BsmBI sites is a dispensable zeocin resistance cassette constitutively expressing the Shigella ble protein. The ble protein sequence, as described above, is shown extending from nucleotide positions 5220 to 5591. The peptide encoding duplexes in Examples 52 and 53 herein below bear cohesive ends complementary to those presented by the vector.

For Examples 3 to 30 for which all are for bacterial expression, cloned peptide sequences are all derived from the annealing of oligonucleotides to create a DNA duplex that is directly ligated into the appropriate vector. Two oligos suffice for Example 20, four are required for all other examples. When the duplex is to be inserted at the N-terminus of Fc (see, Examples 15, 16, 52, and 53 herein) the design is as follows with the ordinal numbers matching the listing of oligos in each example:

When the duplex is to be inserted at the C-terminus of Fc (Examples 3, 4, 5, 10, 11, 12, 13, and 30) the design is as follows:

All remaining examples have the duplex inserted at the C-terminus of Fc and utilize the following design.

No kinasing step is required for the phosphorylation of any of the oligos. A successful insertion of a duplex results in the replacement of the dispensable antibiotic resistance cassette (Zeocin resistance for pAMG21ampR-Pep-Fc and chloramphenicol resistance for pAMG21ampR-Fc-Pep). The resulting change in phenotype is useful for discriminating recombinant from nonrecombinant clones.

The following description gives the uniform method for carrying out the cloning of all 30 bacterially expressed recombinant proteins exemplified herein. Only the set of oligonucleotides and the vector are varied. These specifications are given below in each example.

An oligonucleotide duplex containing the coding region for a given peptide was formed by annealing the oligonucleotides listed in each example. Ten picomoles of each oligo was mixed in a final volume of 10 μl containing 1× ligation buffer along with 0.3 μg of appropriate vector that had been previously digested with restriction endonuclease BsmBI. The mix was heated to 80° C. and allowed to cool at 0.1 degree/sec to room temperature. To this was added 10 μl of 1× ligase buffer plus 400 units of T4 DNA ligase. The sample was incubated at 14 C for 20 min. Ligase was inactivated by heating at 65° C. for 10 minutes. Next, 10 units of restriction endonucleases BsmBI were added followed by incubation at 55 C for one hour to cleave any reformed parental vector molecules. Fifty ul of chemically competent E. coli cells were added and held at 2 C for 20 minutes followed by heat shock at 42 C for 5 second. The entire volume was spread onto Luria Agar plates supplemented with carbenicillin at 200 μg/ml and incubated overnight at 37 C. Colonies were tested for the loss of resistance to the replaceable antibiotic resistance marker. A standard PCR test can be used to confirm the expected size of the duplex insert. Plasmid preparations were obtained and the recombinant insert was verified by DNA sequencing. Half liter cultures of a sequence confirmed construct were grown in Terrific Broth, expression of the peptibody was induced by addition of N-(3-oxo-hexanoyl)-homoserine lactone at 50 ng/ml and after 4-6 hours of shaking at 37 C the cells were centrifuged and the cell paste stored at −20 C.

The following gives for each example the cloning vector and the set of oligonucleotides used for constructing each fusion protein. Also shown is a DNA/protein map.

Bacterial expression of Fc-L-ShK[1-35] inhibitor of Kv1.3. The methods to clone and express the peptibody in bacteria are described above. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ShK[1-35].

Oligos used to form the duplex:

TGGTTCCGGTGGTGGTGGTTCCCGTTCCTG SEQ ID NO: 669 CATCGACACCAT//; CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACA SEQ ID NO: 670 CTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAAC CTGCGGTACCTGC//; CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACA SEQ ID NO: 671 GACGGT//; ATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAAC SEQ ID NO: 672 GGGATTTCGGGATGGTGTCGATGCAGGAACGGGAAC CACCACCACCGGA//;

The oligo duplex is shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCAC   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGGCAAGGACGTAGCTGTGGTAGGGCTTTAGGGCAACGTG  G  S  G  G  G  G  S  R  S  C  I  D  T  I  P  K  S  R  C  T — CGCTTTCCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGG  61 ---------+---------+---------+---------+---------+---------+ 120 GCGAAAGGTCACGTTTGTGAGGTACTTTATGGCAGACAGGAAGACCGCATTTTGGACCCC  A  F  Q  C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G — TACCTGC //SEQ ID NO: 673 121 ------- ATGGACGATTC //SEQ ID NO: 675  T  C — //SEQ ID NO: 674

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of bacterially expressed Fc-L10-ShK(1-35) is further described in Example 38 herein below.

Example 4 Fc-L-ShK[2-35] Bacterial Expression

Bacterial expression of Fc-L-ShK[2-35]. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ShK[2-35].

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCAT SEQ ID NO: 676 CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACA CTCCATGAAAT//; ACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCT SEQ ID NO: 677 GC//; CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACA SEQ ID NO: 678 GACGGTATTTCATGGAGTGTTTGCACTGGAAAGCGG TGCAACGGGA//; TTTCGGGATGGTGTCGATGCAGGAACCACCACCACC SEQ ID NO: 679 GGA//;

The oligo duplex formed is shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCACCGCTTT   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGACGTAGCTGTGGTAGGGCTTTAGGGCAACGTCGCGAAA  G  S  G  G  G  G  S  C  I  D  T  I  P  K  S  R  C  T  A F — CCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTG  61 ---------+---------+---------+---------+---------+---------+ 120 GGTCACGTTTGTGAGGTACTTTATGGCAGACAGGAAGACGGCATTTTGGACGCCATGGAC  Q  C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C — //SEQ ID NO: 681 C //SEQ ID NO: 680 121 — GATTC //SEQ ID NO: 682

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of bacterially expressed Fc-L10-ShK(2-35) is further described in Example 39 herein below.

Example 5 Fc-L-HmK Bacterial Expression

Bacterial expression of Fc-L-HmK. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HmK.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGA SEQ ID NO: 690 CCTGAT//; CCCGGTTTCCGAATGCACCGACATCCGTTGCCGTAC SEQ ID NO: 692 CTCCATGAAATACCGTCTGAACCTGTGCCGTAAAAC CTGCGGTTCCTGC; CTTAGCAGGAACCGCAGGTTTTACGGCACAGGTTCA SEQ ID NO: 693 GACGGT//; ATTTCATGGAGGTACGGCAACGGATGTCGGTGCATT SEQ ID NO: 694 CGGAAACCGGGATCAGGTCTTTGCAGGTACGGGAA CCACCACCACCGGA//

The oligo duplex formed is shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGACCTGATCCCGGTTTCCGAATGCAC   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGGCATGGACGTTTCTGGACTAGCGCCAAAGGCTTACGTG  G  S  G  G  G  G  S  R  T  C  K  D  L  I  P  V  S  E  C  T — CGACATCCGTTGCCGTACCTCCATGAAATACCGTCTCAACCTGTGCCCTAAAACCTGCCG  61 ---------+---------+---------+---------+---------+---------+ 120 GCTGTAGGCAACGGCATGGAGGTACTTTATGGCAGACTTGGACACGGCATTTTGGACGCC  D  I  R  C  R  T  S  M  K  Y  R  L  N  L  C  R  K  T  C  G — //SEQ ID NO: 696 TTCCTGC //SEQ ID NO: 695 121 ------- AAGGACGATTC //SEQ ID NO: 697  S  C —

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 6 Fc-L-KTX1 Bacterial Expression

Bacterial expression of Fc-L-KTX1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-KTX1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAA SEQ ID NO: 698 CGTTAAATGCT//; CCGGTTCCCCGCAGTGCCTGAAACCGTGCAAAGACG SEQ ID NO: 699 CTGGTATGCGTTTCGGTAAATGCATGAACCGTAAAT GCCACTGCACCCCGAAA//; CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGC SEQ ID NO: 700 ATTTACCGAAA//; CGCATACCAGCGTCTTTGCACGGTTTCAGGCACTGC SEQ ID NO: 701 GGGGAACCGGAGCATTTAACGTTGATTTCAACACCG GAACCACCACCACCGGA//;

The oligo duplex formed is shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAACGTTAAATGCTCCGGTTCCCCGCA   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCCACAACTTTAGTTGCAATTTACGAGGCCAAGGGGCGT  G  S  G  G  G  G  S  G  V  E  I  N  V  K  C  S  G  S  P  Q — GTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 CACGGACTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGGCATTTAC  C  L  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  R  K  C — CCACTGCACCCCGAAA //SEQ ID NO: 702 121 ---------+------ GGTGACGTGGGGCTTTATTC //SEQ ID NO: 704  H  C  T  P  K — //SEQ ID NO: 703

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Purification and refolding of Fc-L-KTX1 expressed in bacteria. Frozen, E. coli paste (28 g) was combined with 210 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (4.8 g) was then dissolved in 48 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at 4° C. for 3 days.

The stored refold was then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material was then loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (45 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of PBS and eluted with 100 mM glycine pH 3.0. To the elution peak (2.5 ml), 62.5 μl 2 M tris base was added, and then the pH adjusted material was filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature.

A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 28C). The concentration of the filtered material was determined to be 2.49 mg/ml using a calculated molecular mass of 30,504 g/mol and extinction coefficient of 35,410 M⁻¹cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 28A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 50-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 45 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 28B). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 28D) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.

Purified Fc-L-KTX1 blocked the human Kv1.3 current in a dose-dependent fashion (FIG. 32A and FIG. 32B) by electrophysiology (method was as described in Example 36).

Example 7 Fc-L-HsTx1 Bacterial Expression

Bacterial expression of Fc-L-HsT1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HsTx1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTAC SEQ ID NO: 705 CCCGAAAGAC//; TGCGCTGACCCGTGCCGTAAAGAAACCGGTTGCCCG SEQ ID NO: 706 TACGGTAAATGCATGAACCGTAAATGCAAATGCAAC CGTTGC//; CTTAGCAACGGTTGCATTTGCATTTACGGTTCATGC SEQ ID NO: 707 ATTTACCGTACG//; GGCAACCGGTTTCTTTACGGCACGGGTCAGCGCAGT SEQ ID NO: 708 CTTTCGGGGTACGGCAGGAAGCGGAACCACCACCA CCGGA//;

The duplex formed by the oligos above is shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTACCCCGAAAGACTGCGCTGACCCGTG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCGAAGGACGGCATGGGGCTTTCTGACGCGACTGGGCAC  G  S  G  G  G  G  S  A  S  C  R  T  P  K  D  C  A  D  P  C — CCGTAAAGAAACCGGTTGCCCGTACGGTAAATGCATGAACCGTAAATGCAAATGCAACCG  61 ---------+---------+---------+---------+---------+---------+ 120 GGCATTTCTTTGGCCAACGGGCATGCCATTTACGTACTTGGCATTTACGTTTACGTTGGC  R  K  E  T  G  C  P  Y  G  K  C  M  N  R  K  C  K  C  N  R — TTGC SEQ ID ND: 709 121 ---- 124 AACGATTC SEQ ID NO: 711  C — SEQ ID NO: 710

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 8 Fc-L-MgTx Bacterial Expression

Bacterial expression of Fc-L-MgTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-MgTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGT SEQ ID NO: 712 TAAATGCACCTC//; CCCGAAACAGTGCCTGCCGCCGTGCAAAGCTCAGTT SEQ ID NO: 713 CGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTAA ATGCAAATGCTACCCGCAC//; CTTAGTGCGGGTAGCATTTGCATTTACCGTTCATGC SEQ ID NO: 714 ATTTAGCACCAG//; CGGACTGACCGAACTGAGCTTTGCACGGCGGCAGGC SEQ ID NO: 715 ACTGTTTCGGGGAGGTGCATTTAACGTTGATGATG GTGGAACCACCACCACCGGA//;

The oligos above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGTGGTAGTAGTTGCAATTTACCTCGAGGGGCTTTGTCAC  G  S  G  G  G  G  S  T  I  I  N  V  K  C  T  S  P  K  Q  C — CCTGCCGCCGTGCAAAGCTCAGTTCGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTAA  61 ---------+---------+---------+---------+---------+---------+ 120 GGACGGCGGCACGTTTCGAGTCAAGCCAGTCAGGCGACCACGATTTACGTACTTGCCATT  L  P  P  C  K  A  Q  F  G  Q  S  A  G  A  K  C  M  N  G  K — ATGCAAATGCTACCCGCAC SEQ ID NO: 716 121 ---------+--------- TACGTTTACGATGGGCGTGATTC SEQ ID NO: 718  C  K  C  Y  P  H — SEQ ID NO: 717

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 9 Fc-L-AgTx2 Bacterial Expression

Bacterial expression of Fc-L-AgTx2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-AgTx2.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAA SEQ ID NO: 719 CGTTTCCTGCACCGGT//; TCCCCGCAGTGCATCAAACCGTGCAAAGACGCTGGT SEQ ID NO: 720 ATGCGTTTCGGTAAATGCATGAACCGTAAATGCCAC TGCACCCCGAAA//; CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGC SEQ ID NO: 721 ATTTACCGAAACGCATA//; CCAGCGTCTTTGCACGGTTTGATGCACTGCGGGGAA SEQ ID NO: 722 CCGGTGCAGGAAACGTTGATCGGAACACCGGAACCA CCACCACCGGA//;

The oligos listed above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCGGTTCCCCGCA   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCCACAAGGCTAGTTGCAAAGGACGTGGCCAAGGGGCGT  G  S  G  G  G  G  S  G  V  P  I  N  V  S  C  T  G  S  P  Q — GTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 CACGTAGTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGGCATTTAC  C  I  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  R  K  C — CCACTGCACCCCGAAA_ SEQ ID NO: 723 121 ---------+------ GGTGACGTGGGGCTTTATTC SEQ ID NO: 725  H  C  T  P  K — SEQ ID NO: 724

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Refolding and purification of Fc-L-AgTx2 expressed in bacteria. Frozen, E. coli paste (15 g) was combined with 120 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (4.6 g) was then dissolved in 46 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at −70° C.

The stored refold was defrosted and then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (15 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak (1.5 ml), 70 μl 1 M tris HCl pH 8.5 was added, and then the pH-adjusted material was filtered though a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29C). The concentration of the filtered material was determined to be 1.65 mg/ml using a calculated molecular mass of 30,446 g/mol and extinction coefficient of 35,410 M⁻¹cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 29A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 33-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29D). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μL of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 29E) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.

Example 10 Fc-L-OSK1 Bacterial Expression

Bacterial expression of Fc-L-OSK1. The methods used to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-OSK1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAA SEQ ID NO: 726 CGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACC GTGCAAAAAAG//; CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAAT SEQ ID NO: 727 GCCACTGCACCCCGAAA//; CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGC SEQ ID NO: 728 ATTTACCGAAACGCATACCAGCTTTTTTGCACGGT TCCAGGCACTGA//; CGGGAGATTTTGCATTTAACGTTGATGATAACACCG SEQ ID NO: 729 GAACCACCACCACCGGA//;

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCA   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCCACAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGT  G  S  G  G  G  G  S  G  V  I  I  N  V  K  C  K  I  S  R  Q — GTGCCTGGAACCGTGCAAAAAAGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 CACGGACCTTGGCACGTTTTTTCGACCATACGCAAAGCCATTTACGTACTTGCCATTTAC  C  L  E  P  C  K  K  A  G  M  R  F  G  K  C  M  N  G  K  C — CCACTGCACCCCGAAA SEQ ID NO: 730 121 ---------+------ GGTGACGTGGGGCTTTATTC SEQ ID NO: 732  H  C  T  P  K — SEQ ID NO: 731

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen for later use. Purification of Fc-L10-OSK1 from E. coli paste is described in Example 40 herein below.

Example 11 Fc-L-OSK1(E16K, K20D) Bacterial Expression

Bacterial expression of Fc-L-OSK1(E16K, K20D). The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-OSK1(E16K, K20D).

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAA SEQ ID NO: 733 CGTTAAATGCAAAATCTCCCGTCAGTGCCTGAAAC CGTGCAAAGACG//; CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAAT SEQ ID NO: 734 GCCACTGCACCCCGAAA//; CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGC SEQ ID NO: 735 ATTTACCGAAACGCATACCAGCGTCTTTGCACGGTT TCAGGCACTGA//; CGGGAGATTTTGCATTTAACGTTGATGATAACACCG SEQ ID NO: 736 GAACCACCACCACCGGA//;

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTCCTTCCCCTGTTATCATCAACCTTAAATCCAAAATCTCCCGTCA   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCCACAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGT  G  S  G  G  G  G  S  G  V  I  I  N  V  K  C  K  I  S  R  Q — GTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 CACGGACTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGCCATTTAC  C  L  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  G  K  C — CCACTGCACCCCGAAA SEQ ID NO: 737 121 ---------+------ GGTGACGTGGGGCTTTATTC SEQ ID NO: 739  H  C  T  P  K — SEQ ID NO: 738

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen for later use.

Example 12 Fc-L-Anuroctoxin Bacterial Expression

Bacterial expression of Fc-L-Anuroctoxin. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Anuroctoxin.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGG SEQ ID NO: 740 TCCGCAGCACTGCACCAACTTCTGCCGTAAAAACAA ATGCACCCACG//; GTAAATGCATGAACCGTAAATGCAAATGCTTCAACT SEQ ID NO: 741 GCAAA//; CTTATTTGCAGTTGAAGCATTTGCATTTACGGTTCA SEQ ID NO: 742 TGCATTTACCGTGGGTGCATTTGTTTTTACGGCAG AAGTTGGTGCAG//; TGCTGCGGACCGGTGCATTCTTTGGAACCACCACCA SEQ ID NO: 743 CCGGA//;

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTCCTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCACCAACTTCTG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGTTTCTTACGTGGCCAGGCGTCGTGACGTGGTTGAAGAC  G  S  G  G  G  G  S  K  E  C  T  G  P  Q  H  C  T  N  F  C — CCGTAAAAACAAATGCACCCACGGTAAATGCATGAACCGTAAATGCAAATGCTTCAACTG  61 ---------+---------+---------+---------+---------+---------+ 120 GGCATTTTTGTTTACGTGGGTGCCATTTACGTACTTGGCATTTACGTTTACGAAGTTGAC  R  K  N  K  C  T  H  G  K  C  M  N  R  K  C  K  C  F  N  C — CAAA SEQ ID NO: 744 121 ---- GTTTATTC SEQ ID NO: 746  K — SEQ ID NO: 745

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 13 Fc-L-Noxiustoxin Bacterial Expression

Bacterial expression of Fc-L-Noxiustoxin or Fc-L-NTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-NTX.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGT SEQ ID NO: 747 TAAATGCACCTCCCCGAAACAGTGCTCCAAACCGT GCAAAGAACTGT//; ACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTA SEQ ID NO: 748 AATGCAAATGCTACAACAAC//; CTTAGTTGTTGTAGCATTTGCATTTACCGTTCATGC SEQ ID NO: 749 ATTTAGCACCAGCGGAGGAACCGTACAGTTCTTTG CACGGTTTGGAG//; CACTGTTTCGGGGAGGTGCATTTAACGTTGATGATG SEQ ID NO: 750 GTGGAACCACCACCACCGGA//;

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGTGGTAGTAGTTGCAATTTACGTGGAGGGGCTTTGTCAC  G  S  G  G  G  G  S  T  I  I  N  V  K  C  T  S  P  K  Q  C — CTCCAAACCGTGCAAAGAACTGTACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTAA  61 ---------+---------+---------+---------+---------+---------+ 120 GAGGTTTGGCACGTTTCTTGACATGCCAAGGAGGCGACCACGATTTACGTACTTGCCATT  S  K  P  C  K  E  L  Y  G  S  S  A  G  A  K  C  M  N  G  K — ATGCAAATGCTACAACAAC SEQ ID NO: 751 121 ---------+--------- TACGTTTACGATGTTGTTGATTC SEQ ID NO: 753  C  K  C  Y  N  N — SEQ ID NO: 752

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 14 Fc-L-Pi2 Bacterial Expression

Bacterial expression of Fc-L-Pi2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Pi2.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCAC SEQ ID NO: 754 CAACCCG//; AAACAGTGCTACCCGCACTGCAAAAAAGAAACCGGT SEQ ID NO: 755 TACCCGAACGCTAAATGCATGAACCGTAAATGCA AATGCTTCGGTCGT//; CTTAACGACCGAAGCATTTGCATTTACGGTTCATGC SEQ ID NO: 756 ATTTAGCG//; TTCGGGTAACCGGTTTCTTTTTTGCAGTGCGGGTAG SEQ ID NO: 757 CACTGTTTCGGGTTGGTGCAGGAGATGGTGGAACC ACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCGAAACAGTGCTACCCGCA   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGTGGTAGAGGACGTGGTTGGGCTTTGTCACGATGGGCGT  G  S  G  G  G  G  S  T  I  S  C  T  N  P  K  Q  C  Y  P  H — CTGCAAAAAAGAAACCGGTTACCCGAACGCTAAATGCATGAACCGTAAATGCAAATGCTT  61 ---------+---------+---------+---------+---------+---------+ 120 GACGTTTTTTCTTTGGCCAATGGGCTTGCGATTTACGTACTTGGCATTTACGTTTACGAA  C  K  K  E  T  G  Y  P  N  A  K  C  M  N  R  K  C  K  C  F — CGGTCGT SEQ ID NO: 758 121 ------- GCCAGCAATTC SEQ ID NO: 760  G  R — SEQ ID NO: 759

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 15 ShK[1-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[1-35]-L-Fc. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of ShK[1-35]-L-Fc.

Oligos used to form duplex are shown below:

TATGCGTTCTTGTATTGATACTATTCCAAAA SEQ ID NO: 761 TCTCGTTGTACTGCTTTTCAATGTAAACATT CTATGAAATATCGTCTTTCTT//; TTTGTCGTAAAACTTGTGGTACTTGTTCTGG SEQ ID NO: 762 TGGTGGTGGTTCT//; CACCAGAACCACCACCACCAGAACAAGTAC SEQ ID NO: 763 CACAAGTTTTACGACAAAAAGAAAGACGATA TTTCATAGAATGTTTACATTGA//; AAAGCAGTACAACGAGATTTTGGAATAGTAT SEQ ID NO: 764 CAATACAAGAACG//;

The oligos shown above were used to form the duplex shown below:

TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACA   1 ---------+---------+---------+---------+---------+---------+  60     GCAAGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGT  M  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H — TTCTATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGG  61 ---------+---------+---------+---------+---------+---------+ 120 AAGATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACC  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G — TGGTTCT SEQ ID MO: 765 121 ------- 127 ACCAAGACCAC SEQ ID NO: 767  G  S — SEQ ID NO: 766

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of met-ShK[1-35]-Fc was as described in Example 51 herein below.

Example 16 ShK[2-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[2-35]-L-Fc. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of ShK[2-35]-L-Fc.

Oligos used to form duplex are shown below:

TATGTCTTGTATTGATACTATTCCAAAATCTC SEQ ID NO: 768 GTTGTACTGCTTTTCAATGTAAACATTCTATG AAATATCGTCTTTCTT//; TTTGTCGTAAAACTTGTGGTACTTGTTCTGG SEQ ID NO: 769 TGGTGGTGGTTCT//; CACCAGAACCACCACCACCAGAACAAGTACCA SEQ ID NO: 770 CAAGTTTTACGACAAAAAGAAAGACGATATTT CATAGAATGTTTACATTGA//; AAAGCAGTACAACGAGATTTTGGAATAGTATC SEQ ID NO: 771 AATACAAGA;

The oligos above were used to form the duplex shown below:

TATGTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTC   1 ---------+---------+---------+---------+---------+---------+  60     AGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGTAAG  M  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H  S — TATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGG  61 ---------+---------+---------+---------+---------+---------+ 120 ATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACCACC  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G  G — TTCT SEQ ID NO: 772 121 ---- AAGACCAC SEQ ID NO: 774  S — SEQ ID NO: 773

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of the ShK[2-35]-Fc was as described in Example 50 herein below.

Example 17 Fc-L-ChTx Bacterial Expression

Bacterial expression of Fc-L-ChTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ChTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACC SEQ ID NO: 775 AACGTT//; TCCTGCACCACCTCCAAAGAATGCTGGTCC SEQ ID NO: 776 GTTTGCCAGCGTCTGCACAACACCTCCCGT GGTAAATGCATGAACAAAAAATGCCGTTGC TACTCC//; CTTAGGAGTAGCAACGGCATTTTTTGTTCATG SEQ ID NO: 777 CATTTA//; CCACGGGAGGTGTTGTGCAGACGCTGGCAA SEQ ID NO: 778 ACGGACCAGCATTCTTTGGAGGTGGTGCAGG AAACGTTGGTGAACTGGGAACCACCACCA CCGGA//;

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTGCACCACCTCCAAAGAATG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGGTCAAGTGGTTGCAAAGGACGTGGTGGAGGTTTCTTAC  G  S  G  G  G  G  S  Q  F  T  N  V  S  C  T  T  S  K  E  C — CTGGPCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 GACCAGGCAAACGGTCGCAGACGTGTTGTGGAGGGCACCATTTACGTACTTGTTTTTTAC  W  S  V  C  Q  R  L  H  N  T  S  R  G  K  C  M  N  K  K  C — CCGTTGCTACTCC SEQ ID NO: 779 121 ---------+--- GGCAACGATGAGGATTC SEQ ID NO: 781  R  C  Y  S — SEQ ID NO: 780

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 18 Fc-L-MTX Bacterial Expression

Bacterial expression of Fc-L-MTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-MTX.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCA SEQ ID NO: 782 CCGGT//; TCCAAAGACTGCTACGCTCCGTGCCGTAAAC SEQ ID NO: 783 AGACCGGTTGCCCGAACGCTAAATGCATCAA CAAATCCTGCAAATGCTACGGTTGC//; CTTAGCAACCGTAGCATTTGCAGGATTTGTTG SEQ ID NO: 784 ATGCAT//; TTAGCGTTCGGGCAACCGGTCTGTTTACGG SEQ ID NO: 785 CACGGAGCGTAGCAGTCTTTGGAACCGGTG CAGGAAACGGAACCACCACCACCGGA//;

The oligos above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCACCGGTTCCAAAGACTGCTACGCTCCGTG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCAAAGGACGTGGCCAAGGTTTCTGACGATGCGAGGCAC  G  S  G  G  G  G  S  V  S  C  T  G  S  K  D  C  Y  A  P  C — CCGTAAACAGACCGGTTGCCCGAACGCTAAATGCATCAACAAATCCTGCAAATGCTACGG  61 ---------+---------+---------+---------+---------+---------+ 120 GGCATTTGTCTGGCCAACGGGCTTGCGATTTACGTAGTTGTTTAGGACGTTTACGATGCC  R  K  Q  T  G  C  P  N  A  K  C  I  N  K  S  C  K  C  Y  G — TTGC SEQ ID NO: 786 121 ---- AACGATTC SEQ ID NO: 788  C — SEQ ID NO: 787

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 19 Fc-L-ChTx (K32E) Bacterial Expression

Bacterial expression of Fc-L-ChTx (K32E). The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ChTx (K32E).

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCA SEQ ID NO: 789 ACGTTTCCTG//; CACCACCTCCAAAGAATGCTGGTCCGTTTGCC SEQ ID NO: 790 AGCGTCTGCACAACACCTCCCGTGGTAAATGC ATGAACAAAGAATGCCGTTGCTACTCC//; CTTAGGAGTAGCAACGGCATTCTTTGTTCATG SEQ ID NO: 791 CATTTACCACG//; GGAGGTGTTGTGCAGACGCTGGCAAACGGAC SEQ ID NO: 792 CAGCATTCTTTGGAGGTGGTGCAGGAAACGTT GGTGAACTGGGAACCACCACCACCGGA//;

The oligos shown above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTGCACCACCTCCAAAGAATG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGGTCAAGTGGTTGCAAAGGACGTGGTGGAGGTTTCTTAC  G  S  G  G  G  G  S  Q  F  T  N  V  S  C  T  T  S  K  E  C — CTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAGAATG  61 ---------+---------+---------+---------+---------+---------+ 120 GACCAGGCAAACGGTCGCAGACGTGTTGTGGAGGGCACCATTTACGTACTTGTTTCTTAC  W  S  V  C  Q  R  L  H  N  T  S  R  G  K  C  M  N  K  E  C — CCGTTGCTACTCC SEQ ID NO: 793 121 ---------+--- GGCAACGATGAGGATTC SEQ ID NO: 795  R  C  Y  S — SEQ ID NO: 794

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 20 Fc-L-Apamin Bacterial Expression

Bacterial expression of Fc-L-Apamin. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Apamin.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCA SEQ ID NO: 796 AAGCTCCGGAAACCGCTCTGTGCGCTCGTCG TTGCCAGCAGCACGGT//; CTTAACCGTGCTGCTGGCAACGACGAGCGCA SEQ ID NO: 797 CAGAGCGGTTTCCGGAGCTTTGCAGTTGCAGG AACCACCACCACCGGA//;

The oligos above were used to form the duplex shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCAAAGCTCCGGAAACCGCTCTGTGCGCTCG  1 ---------+---------+---------+---------+---------+---------+ 60     AGGCCACCACCACCAAGGACGTTGACGTTTCGAGGCCTTTGGCGAGACACGCGAGC  G  S  G  G  G  G  S  C  N  C  K  A  P  E  T  A  L  C  A  R — TCGTTGCCAGCAGCACGGT SEQ ID NO: 798 61 ---------+--------- AGCAACGGTCGTCGTGCCAATTC SEQ ID NO: 800  R  C  Q  Q  H  G — SEQ ID NO: 799

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 21 Fc-L-Scyllatoxin Bacterial Expression

Bacterial expression of Fc-L-Scyllatoxin or Fc-L-ScyTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ScyTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGC SEQ ID NO: 801 AACCTGCG//; TATGTGCCAGCTGTCCTGCCGTTCCCTGGGTC SEQ ID NO: 802 TGCTGGGTAAATGCATCGGTGACAAATGCGAA TGCGTTAAACAC//; CTTAGTGTTTAACGCATTCGCATTTGTCACCG SEQ ID NO: 803 ATGCATTT//; ACCCAGCAGACCCAGGGAACGGCAGGACA SEQ ID NO: 804 GCTGGCACATACGCAGGTTGCAGAAAGCGG AACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCAACCTGCGTATGTGCCAGCTGTCCTGCCG  1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCGAAAGACGTTGGACGCATACACGGTCGACAGGACGGC  G  S  G  G  G  G  S  A  F  C  N  L  R  M  C  Q  L  S  C  R — TTCCCTGGGTCTGCTGGGTAAATGCATCGGTGACAAATGCGAATGCGTTAAACAC SEQ ID NO: 805 61 ---------+---------+---------+---------+---------+---------+ AAGGGACCCAGACGACCCATTTACGTAGCCACTGTTTACGCTTACGCAATTTGTGATTC SEQ ID NO: 807  S  L  G  L  L  G  K  C  I  G  D  K  C  E  C  V  K  H — SEQ ID NO: 806

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 22 Fc-L-IbTx Bacterial Expression

Bacterial expression of Fc-L-lbTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-lbTx.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACC SEQ ID NO: 808 GACGTTGACTGCTCCGT//; TTCCAAAGAATGCTGGTCCGTTTGCAAAGACC SEQ ID NO: 809 TGTTCGGTGTTGACCGTGGTAAATGCATGGGT AAAAAATGCCGTTGCTACCAG//; CTTACTGGTAGCAACGGCATTTTTTACCCATG SEQ ID NO: 810 CATTTACCACGGTCAA//; CACCGAACAGGTCTTTGCAAACGGACCAGCA SEQ ID NO: 811 TTCTTTGGAAACGGAGCAGTCAACGTCGGTGA ACTGGGAACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCGACGTTGACTGCTCCGTTTCCAAAGAATG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCGAGGAGCACCAAGGGTCAAGTGGCTGCAACTGACGAGGCAAAGGTTTCTTAC  G  S  G  G  G  G  S  Q  F  T  D  V  D  C  S  V  S  K  E  C — CTGGTCCGTTTGCAAAGACCTGTTCGGTGTTGACCGTGGTAAATGCATGGGTAAAAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 GACCAGGCAAACGTTTCTGGACAAGCCACAACTGGCACCATTTACGTACCCATTTTTTAC  W  S  V  C  K  D  L  F  G  V  D  R  G  K  C  M  G  K  K  C — CCGTTGCTACCAG SEQ ID NO: 812 121 ---------+--- GGCAACGATGGTCATTC SEQ ID NO: 814  R  C  Y  Q — SEQ ID NO: 813

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 23 Fc-L-HaTx1 Bacterial Expression

Bacterial expression of Fc-L-HaTx1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HaTx1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGT SEQ ID NO: 815 TACCTGTTCGGTGGTTG//; CAAAACCACCTCCGACTGCTGCAAACACCTGG SEQ ID NO: 816 GTTGCAAATTCCGTGACAAATACTGCGCTTGG GACTTCACCTTCTCC//; CTTAGGAGAAGGTGAAGTCCCAAGCGCAGTA SEQ ID NO: 817 TTTGTCACGGAATTTGC//; AACCCAGGTGTTTGCAGCAGTCGGAGGTGGT SEQ ID NO: 818 TTTGCAACCACCGAACAGGTAACGGCATTCG GAACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACCTGTTCGGTGGTTGCAAAACCACCTC   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCTTACGGCAATGGACAAGCCACCAACGTTTTGGTGGAG  G  S  G  G  G  G  S  E  C  R  Y  L  F  G  G  C  K  T  T  S — CGACTGCTGCAAACACCTGGGTTGCAAATTCCGTGACAAATACTGCGCTTGGGACTTCAC  61 ---------+---------+---------+---------+---------+---------+ 120 GCTGACGACGTTTGTGGACCCAACGTTTAAGGCACTGTTTATGACGCGAACCCTGAAGTG  D  C  C  K  H  L  G  C  K  F  R  D  K  Y  C  A  W  D  F  T — CTTCTCC SEQ ID NO: 819 121 ------- GAAGAGGATTC SEQ ID NO: 821  F  S — SEQ ID NO: 820

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Refolding and purification of Fc-L-HaTx1 expressed in bacteria. Frozen, E. coli paste (13 g) was combined with 100 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (2.6 g) was then dissolved in 26 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at −70° C.

The stored refold was defrosted and then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H₃PO₄. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (15 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak (1.4 ml), 70 μl 1 M tris HCl pH 8.5 was added, and then the pH adjusted material was filtered though a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29F). The concentration of the filtered material was determined to be 1.44 mg/ml using a calculated molecular mass of 30,469 g/mol and extinction coefficient of 43,890 M⁻¹ cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 29B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 33-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29G). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 29H) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.

Example 24 Fc-L-PaTx2 Bacterial Expression

Bacterial expression of Fc-L-PaTx2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-PaTx2.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGA SEQ ID NO: 822 AATGGA//; TGTGGACCTGCGACGAAGAACGTAAATGCTG SEQ ID NO: 823 CGAAGGTCTGGTTTGCCGTCTGTGGTGCAAAC GTATCATCAACATG//; CTTACATGTTGATGATACGTTTGCACCACAGA SEQ ID NO: 824 CGGCAAA//; CCAGACCTTCGCAGCATTTACGTTCTTCGTCG SEQ ID NO: 825 CAGGTCCACATCCATTTCTGGCAGTAGGAACC ACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGATGTGGACCTGCGACGAAGAACG  1 ---------+---------+---------+---------+---------+---------+ 60     AGGCCACCACCACCAAGGATGACGGTCTTTACCTACACCTGGACGCTGCTTCTTGC  G  S  G  G  G  G  S  Y  C  Q  K  W  M  W  T  C  D  E  E  R — TAAATGCTGCGAAGGTCTGGTTTGCCGTCTGTGGTGCAAACGTATCATCAACATG SEQ ID NO: 826 61 ---------+---------+---------+---------+---------+----- ATTTACGACGCTTCCAGACCAAACGGCAGACACCACGTTTGCATAGTAGTTGTACATTC SEQ ID NO: 828  K  C  C  E  G  L  V  C  R  L  W  C  K  R  I  I  N  M — SEQ ID NO: 827

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 25 Fc-L-wGVIA Bacterial Expression

Bacterial expression of Fc-L-wGVIA. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-wGVIA.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCC SEQ ID NO: 829 CGGGTT//; CCTCCTGCTCCCCGACCTCCTACAACTGCTGC SEQ ID NO: 830 CGTTCCTGCAACCCGTACACCAAACGTTGCTA CGGT; CTTAACCGTAGCAACGTTTGGTGTACGGGTTG SEQ ID NO: 831 CAGGAA//; CGGCAGCAGTTGTAGGAGGTCGGGGAGCAGGA SEQ ID NO: 832 GGAACCCGGGGATTTGCAGGAACCACCACCAC CGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTTCCTCCTGCTCCCCGACCTCCTA  1 ---------+---------+---------+---------+---------+---------+ 60     AGGCCACCACCACCAAGGACGTTTAGGGGCCCAAGGAGGACGAGGGGCTGGAGGAT  G  S  G  G  G  G  S  C  K  S  P  G  S  S  C  S  P  T  S  Y — CAACTGCTGCCGTTCCTGCAACCCGTACACCAAACGTTGCTACGGT SEQ ID NO: 833 61 ---------+---------+---------+---------+------ GTTGACGACGGCAAGGACGTTGGGCATGTGGTTTGCAACGATGCCAATTC SEQ ID NO: 835  N  C  C  R  S  C  N  P  Y  T  K  R  C  Y  G SEQ ID NO: 834

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 26 Fc-L-ωMVIIA Bacterial Expression

Bacterial expression of Fc-L-ωMVIIA. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ωMVIIA.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTA SEQ ID NO: 836 AA//; GGTGCTAAATGCTCCCGTCTGATGTACGACTG SEQ ID NO: 837 CTGCACCGGTTCCTGCCGTTCCGGTAAATGCG GT//; CTTAACCGCATTTACCGGAACGGCAGGAACCG SEQ ID NO: 838 GT//; GCAGCAGTCGTACATCAGACGGGAGCATTTAG SEQ ID NO: 839 CACCTTTACCTTTGCAGGAACCACCACCACCG GA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAAGGTGCTAAATGCTCCCGTCTGATGTA 1 ---------+---------+---------+---------+---------+---------+ 60     AGGCCACCACCACCAAGGACGTTTCCATTTCCACGATTTACGAGGGCAGACTACAT  G  S  G  G  G  G  S  C  K  G  K  G  A  K  C  S  R  L  M  Y — CGACTGCTGCACCGGTTCCTGCCGTTCCGGTAAATGCGGT SEQ ID NO: 840 61 ---------+---------+---------+---------+ GCTGACGACGTGGCCAAGGACGGCAAGGCCATTTACGCCAATTC SEQ ID NO: 842  D  C  C  T  G  S  C  R  S  G  K  C  G — SEQ ID NO: 841

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 27 Fc-L-PtuI Bacterial Expression

Bacterial expression of Fc-L-Ptu1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Ptu1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAG SEQ ID NO: 843 ACTGCATC//; GCTCCGGGTGCTCCGTGCTTCGGTACCGACA SEQ ID NO: 844 AACCGTGCTGCAACCCGCGTGCTTGGTGCTCC TCCTACGCTAACAAATGCCTG//; CTTACAGGCATTTGTTAGCGTAGGAGGAGCAC SEQ ID NO: 845 CAAGCACG//; CGGGTTGCAGCACGGTTTGTCGGTACCGAAGC SEQ ID NO: 846 ACGGAGCACCCGGAGCGATGCAGTCTTTTTCA GCGGAACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATCGCTCCGGGTGCTCCGTGCTT   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCGACTTTTTCTGACGTAGCGAGGCCCACGAGGCACGAA  G  S  G  G  G  G  S  A  E  K  D  C  I  A  P  G  A  P  C  F — CGGTACCGACAAACCGTGCTGCAACCCGCGTGCTTGGTGCTCCTCCTACGCTAACAAATG  61 ---------+---------+---------+---------+---------+---------+ 120 GCCATGGCTGTTTGGCACGACGTTGGGCGCACGAACCACGAGGAGGATGCGATTGTTTAC  G  T  D  K  P  C  C  N  P  R  A  W  C  S  S  Y  A  N  K  C — CCTG SEQ ID NO: 847 121 ---- GGACATTC SEQ ID NO: 849  L — SEQ ID NO: 648

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 28 Fc-L-ProTx1 Bacterial Expression

Bacterial expression of Fc-L-ProTx1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ProTx1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGT SEQ ID NO: 850 TACTGGCTGG//; GTGGTTGCTCCGCTGGTCAGACCTGCTGCAA SEQ ID NO: 851 ACACCTGGTTTGCTCCCGTCGTCACGGTTGG TGCGTTTGGGACGGTACCTTCTCC//; CTTAGGAGAAGGTACCGTCCCAAACGCACC SEQ ID NO: 852 AACCGTGACGA//; CGGGAGCAAACCAGGTGTTTGCAGCAGGTCT SEQ ID NO: 853 GACCAGCGGAGCAACCACCCAGCCAGTAAC GGCATTCGGAACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGGGTGGTTGCTCCGCTGGTCA   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGCTTACGGCAATGACCGAGCCACCAACGAGGCGAGCAGT  G  S  G  G  G  G  S  E  C  R  Y  W  L  G  G  C  S  A  G  Q — GACCTGCTGCAAACACCTGGTTTGCTCCCGTCGTCACGGTTGGTGCGTTTGGGACGGTAC  61 ---------+---------+---------+---------+---------+---------+ 120 CTGGACGACGTTTGTGGACCAAACGAGGGCAGCAGTGCCAACCACGCAAACCCTGCCATG  T  C  C  K  H  L  V  C  S  R  R  H  G  W  C  V  W  D  G  T — CTTCTCC SEQ ID NO: 854 121 -------> GAAGAGGATTC SEQ ID NO: 856  F  S — SEQ ID NO: 855

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 29 Fc-L-BeKM1 Bacterial Expression

Bacterial expression of Fc-L-BeKM1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-BeKM1.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCG SEQ ID NO: 857 ACATCAAATG//; CTCCGAATCCTACCAGTGCTTCCCGGTTTGCA SEQ ID NO: 858 AATCCCGTTTCGGTAAAACCAACGGTCGTTGC GTTAACGGTTTCTGCGACTGCTTC//; CTTAGAAGCAGTCGCAGAAACCGTTAACGCA SEQ ID NO: 859 ACGACCGTTGG//; TTTTACCGAAACGGGATTTGCAAACCGGGAAG SEQ ID NO: 860 CACTGGTAGGATTCGGAGCATTTGATGTCGGT CGGACGGGAACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATGCTCCGAATCCTACCAGTG   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGGCAGGCTGGCTGTAGTTTACGAGGCTTAGGATGGTCAC  G  S  G  G  G  G  S  R  P  T  D  I  K  C  S  E  S  Y  Q  C — CTTCCCGGTTTGCAAATCCCGTTTCGGTAAAACCAACGGTCGTTGCGTTAACGGTTTCTG  61 ---------+---------+---------+---------+---------+---------+ 120 GAAGGGCCAAACGTTTAGGGCAAAGCCATTTTGGTTGCCAGCAACGCAATTGCCAAAGAC  F  P  V  C  K  S  R  F  G  K  T  N  G  R  C  V  N  G  F  C — CGACTGCTTC SEQ ID NO: 861 121 ---------+ GCTGACGAAGATTC SEQ ID NO: 863  D  C  F — SEQ ID NO: 862

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 30 Fc-L-CTX Bacterial Expression

Bacterial expression of Fc-L-CTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-CTX.

Oligos used to form duplex are shown below:

TGGTTCCGGTGGTGGTGGTTCCATG SEQ ID NO: 864 TGCATGCCGTGCTTCAC//; CACCGACCACCAGATGGCTCGTAAATGCG SEQ ID NO: 865 ACGACTGCTGCGGTGGTAAAGG TCGTGGTAAATGCTACGGTCCG CAGTGCCTGTGCCGT//; CTTAACGGCACAGGCACTGCGGACCG SEQ ID NO: 866 TAGCATTTACCACGAC//; CTTTACCACCGCAGCAGTCGTCGCATTTA SEQ ID NO: 867 CGAGCCATCTGGTGGTCGGTGG TGAAGCACGGCATGCACATGGA ACCACCACCACCGGA//;

The oligos above were used to form the duplex below:

TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCACCACCGACCACCAGATGGC   1 ---------+---------+---------+---------+---------+---------+  60     AGGCCACCACCACCAAGGTACACGTACGGCACGAAGTGGTGGCTGGTGGTCTACCG  G  S  G  G  G  G  S  M  C  M  P  C  F  T  T  D  H  Q  M  A — TCGTAAATGCGACGACTGCTGCGGTGGTAAAGGTCGTGGTAAATGCTACCGTCCGCAGTG  61 ---------+---------+---------+---------+---------+---------+ 120 AGCATTTACGCTGCTGACGACGCCACCATTTCCAGCACCATTTACGATGCCAGGCGTCAC  R  K  C  D  D  C  C  G  G  K  G  R  G  K  C  Y  G  P  Q  C — CCTGTGCCGT SEQ ID NO: 868 121 ---------+ GGACACGGCACCAC SEQ ID NO: 870  L  C  R — SEQ ID NO: 869

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 31 N-Terminally PEGylated-Des-Arg1-ShK

Peptide Synthesis of reduced Des-Arq1-ShK. Des-Arg1-ShK, having the sequence SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC  (Peptide 1, SEQ ID NO: 92) was synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHB Fmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids were purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka. The following side-chain protection strategy was employed: Asp(O^(t)Bu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu), Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(^(ψMe,Me)Pro)-OH and Fmoc-Leu-Ser(^(ψMe,Me)Pro)-OH, were used in the chain assembly and were obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids were activated with 20 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling were carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions were carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following synthesis, the resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected and released from the resin by treatment with a TFA/EDT/TIS/H₂O (92.5:2.5:2.5:2.5 (v/v)) solution at room temperature for 1 hour. The volatiles were then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The crude peptide was then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-Sciex™ API Electro-spray mass spectrometer was used to confirm correct peptide product mass. Crude peptide was obtained in 143 mg yield at approximately 70% pure based as estimated by analytical RP-HPLC analysis. Reduced Des-Arg1-ShK (Peptide 1) Retention time (Rt)=5.31 minutes, calculated molecular weight=3904.6917 Da (average); Experimental observed molecular weight 3907.0 Da.

Folding of Des-Arq1-ShK (Disulphide bond formation). Following TFA cleavage and peptide precipitation, reduced Des-Arg1-ShK was then air-oxidized to give the folded peptide. The crude cleaved peptide was extracted using 20% AcOH in water (v/v) and then diluted with water to a concentration of approximately 0.15 mg reduced Des-Arg1-ShK per mL, the pH adjusted to about 8.0 using NH₄OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process was monitored by LC-MS analysis. Following this, folded Des-Arg1-ShK peptide was purified using reversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded Des-Arg1-ShK crude peptide eluted earlier (when compared to the elution time in its reduced form) at approximately 25% buffer B. Folded Des-Arg1-ShK (Peptide 2) was obtained in 23.2 mg yield in >97% purity as estimated by analytical RP-HPLC analysis (FIG. 20A). Calculated molecular weight=3895.7693 Da (monoisotopic), experimental observed molecular weight=3896.5 Da(analyzed on a Waters LCT Premier Micromass MS Technologies). (FIG. 20B). Des-Arg1-ShK disulfide connectivity was C1-C6, C2-C4, C3-C5.

N-terminal PEGylation of Folded Des-Arq1-ShK. Folded Des-Arg1-ShK, (Peptide 2) was dissolved in water at 1 mg/ml concentration. A 2 M MeO-PEG-Aldehyde, CH₃O—[CH₂CH₂O]n-CH₂CH₂CHO (average molecular weight 20 kDa), solution in 50 mM NaOAc, pH 4.5, and a separate 1 M solution of NaCNBH₃ were freshly prepared. The peptide solution was then added to the MeO-PEG-Aldehyde containing solution and was followed by the addition of the NaCNBH₃ solution. The reaction stoichiometry was peptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction was left for 48 hours, and was analyzed on an Agilent 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated folded Des-Arg1-ShK constituted approximately 58% of the crude product by analytical RP-HPLC. Mono Pegylated Des-Arg1-ShK was then isolated using a HiTrap™ 5 ml SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions were analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC (as described for the crude). SDS-PAGE gels were run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product was then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product was then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product. N-Terminally PEGylated-Des-Arg1-ShK (Peptide 3) was isolated in 1.7 mg yield with 85% purity as estimated by analytical RP-HPLC analysis (FIG. 23).

The N-Terminally PEGylated-Des-Arg1-ShK, also referred to as “PEG-ShK[2-35]”, was active in blocking human Kv1.3 (FIG. 38A and FIG. 38B) as determined by patch clamp electrophysiology (Example 36).

Example 32 N-Terminally PEGylated ShK

The experimental procedures of this working example correspond to the results shown in FIG. 17.

Peptide Synthesis of reduced ShK. ShK, having the amino acid sequence

RSCIDTIPKSRCTAFQCKHSMKY (Peptide 4, SEQ ID NO: 5) RLSFCRKTCGTC was synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHB Fmoc-Cys(Trt)-resin. N-alpha-9-fluorenylmethyloxycarbonyl) and side-chain protected amino acids were purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka. The following side-chain protection strategy was employed: Asp(O^(t)Bu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu), Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(^(ψMe,Me)Pro)-OH and Fmoc-Leu-Ser(^(ψMe,Me)Pro)-OH, were used in the chain assembly and were obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids were activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling were carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotections were carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following synthesis, the resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected and released from the resin by treatment with a TFA/EDT/TIS/H₂O (92.5:2.5:2.5:2.5 (v/v)) solution at room temperature for 1 hour. The volatiles were then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The crude peptide was then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-Sciex API Electro-spray mass spectrometer was used to confirm correct peptide product mass. Crude peptide was approximately was obtained 170 mg yield at about 45% purity as estimated by analytical RP-HPLC analysis. Reduced ShK (Peptide 4) Retention time (Rt)=5.054 minutes, calculated molecular weight=4060.8793 Da (average); experimental observed molecular weight=4063.0 Da.

Folding of ShK (Disulphide bond formation). Following TFA cleavage and peptide precipitation, reduced ShK was then air oxidized to give the folded peptide. The crude cleaved peptide was extracted using 20% AcOH in water (v/v) and then diluted with water to a concentration of approximately 0.15 mg reduced ShK per mL, the pH adjusted to about 8.0 using NH₄OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process was monitored by LC-MS analysis. Following this, folded ShK peptide was purified by reversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded ShK crude peptide eluted earlier (when compared to the elution time in its reduced form) at approximately 25% buffer B. Folded ShK (Peptide 5) was obtained in 25.5 mg yield in >97% purity as estimated by analytical RP-HPLC analysis. See FIG. 60. Calculated molecular weight=4051.8764 Da (monoisotopic); experimental observed molecular weight=4052.5 Da (analyzed on Waters LCT Premier Micromass MS Technologies). ShK disulfide connectivity was C1-C6, C2-C4, and C3-C5.

N-terminal PEGylation of Folded ShK. Folded ShK, having the amino acid sequence

RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 5) can be dissolved in water at 1 mg/ml concentration. A 2 M MeO-PEG-Aldehyde, CH₃O—CH₂CH₂O]n-CH₂CH₂CHO (average molecular weight 20 kDa), solution in 50 mM NaOAc, pH 4.5 and a separate 1 M solution of NaCNBH₃ can be freshly prepared. The peptide solution can be then added to the MeO-PEG-Aldehyde containing solution and can be followed by the addition of the NaCNBH₃ solution. The reaction stoichiometry can be peptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction can be left for 48 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated Shk (Peptide 6) can be then isolated using a HiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM sodium acetate, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.

Example 33 N-Terminally PEGylated ShK by Oxime Formation

Peptide Synthesis of reduced ShK. ShK, having the sequence

RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (SEQ ID NO: 5) can be synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHB Fmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids can be purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-Tentagel™ resin can be purchased from Fluka. The following side-chain protection strategy can be employed: Asp(O^(t)Bu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu), Thr(O^(t)Bu) and Tyr(O^(t)Bu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(Ψ^(Me,Me)Pro)-OH and Fmoc-Leu-Ser(ψ^(Me,Me)Pro)-OH, can be used in the chain assembly and can be obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions can be carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following the chain-assembly of the Shk peptide, Boc-amionooxyacetic acid (1.2 equiv) can be coupled at the N-terminus using 0.5 M HBTU in DMF with 4 equiv collidine for 5 minutes. Following synthesis, the resin can be then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin can be deprotected and released from the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O (90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. The volatiles can be then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The aminooxy-Shk peptide (Peptide 7) can be then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phase high-performance liquid chromatography can be performed on C18, 5 μm, 2.2 cm×25 cm) column. Chromatographic separations can be achieved using linear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA and 0.1% aminooxyacetic acid), typically 5-95% over 90 minutes at 15 mL/min. Preparative HPLC fractions can be characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized.

N-Terminal PEGylation of Shk by Oxime Formation. Lyophilized aminooxy-Shk (Peptide 7) can be dissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molar excess of MeO-PEG-Aldehyde, CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CHO (average molecular weight 20 kDa). The reaction can be left for 24 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated reduced Shk constituted approximately 58% of the crude product by analytical RP-HPLC. Mono PEGylated (oximated) Shk (Peptide 8) can be then isolated using a HiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A 20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.

Folding of ShK (Disulphide bond formation). The mono-PEGylated (oximated) Shk can be dissolved in 20% AcOH in water (v/v) and can be then diluted with water to a concentration of approximately 0.15 mg peptide mL, the pH adjusted to about 8.0 using NH₄OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process can be monitored by LC-MS analysis. Following this, folded mono-PEGylated (oximated) Shk (Peptide 9) can be purified using by reversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated) ShK disulfide connectivity can be C1-C6, C2-C4, and C3-C5.

Example 34 N-Terminally PEGylated ShK (Amidation)

The experimental procedures of this working example correspond to the results shown in FIG. 18.

N-Terminal PEGylation of Shk by Amide Formation. A 10 mg/mL solution of folded Shk (Peptide 5), in 100 mM Bicine pH 8.0, can be added to solid succinimidyl ester of 20 kDa PEG propionic acid (mPEG-SPA; CH₃O—[CH₂CH₂O]n-CH₂CH₂CO—NHS) at room temperature using a 1.5 molar excess of the mPEG-SPA to Shk. After one hour with gentle stirring, the mixture can be diluted to 2 mg/mL with water, and the pH can be adjusted to 4.0 with dilute HCl. The extent of mono-pegylated Shk (Peptide 10), some di-PEGylated Shk or tri-PEGylated Shk, unmodified Shk and succinimidyl ester hydrolysis can be determined by SEC HPLC using a Superdex™ 75 HR 10/30 column (Amersham) eluted with 0.05 M NaH₂PO₄, 0.05 M Na₂HPO₄, 0.15 M NaCl, 0.01 M NaN₃, pH 6.8, at 1 mL/min. The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed N-terminally PEGylated (amidated) ShK (Peptide 10) can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.

Example 35 Fc-L-SmIIIA

Fc-SmIIIA expression vector. A 104 bp BamHI-NotI fragment containing partial linker sequence and SmIIIA peptide encoded with human high frequency codons was assembled by PCR with overlapping primers 3654-50 and 3654-51 and cloned into to the 7.1 kb NotI-BamHI back bone to generate pcDNA3.1(+) CMVi-hFc-SmIIIA as described in Example 1.

  BamHI 5′GGATCCGGAGGAGGAGGAAGCTGCTGCAACGGC SEQ ID NO: 872 CGCCGCGGCTGCAGCAGCCGCTGG                        C  C  N  G  R SEQ ID NO: 873 R  G  C  S  S  R  W TGCCGCGACCACAGCCGC C  R  D  H  S  R  C TGCTGCTGAGCGGCCGC3′// C     NotI Forward 5′-3′: GGAGGAGGATCCGGAGGAGGAGGAAGCTGCTG SEQ ID NO: 874 CAACGGCCGCCGCGGCTGCAGCAGC CGC// Reverse 5′-3′: ATTATTGCGGCCGCTCAGCAGCAGCGGCTGTG SEQ ID NO: 875 GTCGCGGCACCAGCGGCTGCTGCAG CCGC The sequences of the BamHI to NotI fragments in the final constructs were verified by sequencing.

Transient expression of Fc-L-SmIIIa. 7.5 ug of the toxin peptide Fc fusion construct pcDNA3.1(+) CMVi-hFc-SmIIIA were transfected into 293-T cells in 10 cm tissue culture plate with FuGENE 6 as transfection reagent. Culture medium was replaced with serum-free medium at 24 hours post-transfection and the conditioned medium was harvested at day 5 post-transfection. Transient expression of Fc-SmIIIA from 293-T cells was analyzed by Western blot probed with anti-hFc antibody (FIG. 25A and FIG. 25B). Single band of expressed protein with estimated MW was shown in both reduced and non-reduced samples. Transient expression level of Fc-SmIIIA was further determined to be 73.4 μg/ml according to ELISA.

Example 36 Electrophysiology Experiments

Cell Culture. Stable cell line expressing human Kv1.3 channel was licensed from Biofocus. Cells were kept at 37° C. in 5% CO₂ environment. Culture medium contains DMEM with GlutaMax™ (Invitrogen), 1× non-essential amino acid, 10% fetal bovine serum and 500 μg/mL geneticin. Cells were plated and grown at low confluence on 35 mm culture dishes for at least 24 hours prior to electrophysiology experiments.

Electrophysiology Recording by Patch Clamping. Whole-cell currents were recorded from single cells by using tight seal configuration of the patch-clamp technique. A 35 mm culture dish was transferred to the recording stage after rinsing and replacing the culture medium with recording buffer containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, and 5 mM Glucose. pH was adjusted to 7.4 with NaOH and the osmolarity was set at 300 mOsm. Cells were perfused continuously with the recording buffer via one of the glass capillaries arranged in parallel and attached to a motorized rod, which places the glass capillary directly on top of the cell being recorded. Recording pipette solution contained 90 mM K-gluconate, 20 mM KF, 10 mM NaCl, 1 mM MgCl₂-6H₂O, 10 mM EGTA, 5 mM K2-ATP, and 10 mM HEPES. The pH for the internal solution was adjusted to 7.4 with KOH and the osmolarity was set at 280 mOsm. Experiments were performed at room temperature (20-22° C.) and recorded using Multiclamp™ 700 A amplifier (Molecular Devices Inc.). Pipette resistances were typically 2-3 MΩ.

Protein toxin potency determination on Kv1.3 current: HEK293 cells stably expressing human Kv1.3 channel were voltage clamped at −80 mV holding potential. Outward Kv1.3 currents were activated by giving 200 msec long depolarizing steps to +30 mV from the holding potential of −80 mV and filtered at 3 kHz. Each depolarizing step was separated from the subsequent one with a 10 s interval. Analogue signals were digitized by Digidata™ 1322A digitizer (Molecular Devices) and subsequently stored on computer disk for offline analyses using Clampfit™ 9 (Molecular Devices Inc.). In all studies, stable baseline K_(V)1.3 current amplitudes were established for 4 minutes before starting the perfusion of the protein toxin at incremental concentrations. A steady state block was always achieved before starting the perfusion of the subsequent concentration of the protein toxin.

Data analysis. Percent of control (POC) is calculated based on the following equation: (K_(V)1.3 current after protein toxin addition/K_(V)1.3 current in control)*100. At least 5 concentrations of the protein toxin (e.g. 0.003, 0.01, 0.03, 0.1, 0.3, 100 nM) were used to calculate the IC₅₀ value. IC₅₀ values and curve fits were estimated using the four parameter logistic fit of XLfit software (Microsoft Corp.). IC₅₀ values are presented as mean value±s.e.m. (standard error of the mean).

Drug preparations. Protein toxins (typically 10-100 μM) were dissolved in distilled water and kept frozen at −80° C. Serial dilutions of the stock protein toxins were mixed into the recording buffer containing 0.1% bovine serum albumin (BSA) and subsequently transferred to glass perfusion reservoirs. Electronic pinch valves controlled the flow of the protein toxin from the reservoirs onto the cell being recorded.

Example 37 Immunobiology and Channel Binding

Inhibition of T cell cytokine Production following PMA and anti-CD3 antibody stimulation of PBMCs. PBMC's were previously isolated from normal human donor Leukophoresis packs, purified by density gradient centrifugation (Ficoll Hypaque), cryopreserved in CPZ Cryopreservation Medium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's were thawed (95% viability), washed, and seeded at 2×10⁵ cells per well in culture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin 2 mM L-glutamine, 100 uM non-essential amino acids, and 20 uM 2-ME) in 96-well flat-bottom tissue culture plates. Cells were pre-incubated with serially diluted (100 nM-0.001 nM final) ShK[1-35], Fc-L10-ShK[1-35] or fc control for 90 min before stimulating for 48 hr with PMA/anti-CD3 (1 ng/ml and 50 ng/ml, respectively) in a final assay volume of 200 ul. Analysis of the assay samples was performed using the Meso Scale Discovery (MSD) SECTOR™ Imager 6000 (Meso Scale Discovery, Gaithersbury, Md.) to measure the IL-2 and IFNg protein levels by utilizing electrochemiluminescence (ECL). The conditioned medium (50 ul) was added to the MSD Multi-spot 96-well plates (each well containing three capture antibodies; IL-2, TNF, IFNγ). The plates were sealed, wrapped in tin foil, and incubated at room temperature on a plate shaker for 2 hr. The wells were washed 1× with 200 ul PBST (BIOTEK, Elx405 Auto Plate Washer). For each well, 20 ul of Ruthenium-labeled detection antibodies (1 μg/ml final in Antibody Dilution Buffer; IL-1, TNF, IFNγ) and 130 ul of 2×MSD Read Buffer added, final volume 150 ul. The plates were sealed, wrapped in tin foil, and incubated at room temperature on a plate shaker for 1 hr. The plates were then read on the SECTOR™ Imager 6000. FIGS. 35A & 35B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg production from T cells in a dose-dependent manner. Compared to native ShK[1-35] peptide, the peptibody produces a greater extent of inhibition (POC=Percent Of Control of response in the absence of inhibitor).

Inhibition of T cell cytokine production following anti-CD3 and anti-CD28 antibody stimulation of PBMCs. PBMCs were previously isolated from normal human donor Leukopheresis packs, purified by density gradient centrifugation (Ficoll Hypaque), and cryopreserved using INCELL Freezing Medium. PBMCs were thawed (95% viability), washed, and seeded (in RPMI complete medium containing serum replacement, PSG) at 2×10⁵ cells per well into 96-well flat bottom plates. Cells were pre-incubated with serially diluted (100 nM-0.003 nM final) ShK[1-35], Fc-L10-ShK[1-35], or Fc control for 1 hour before the addition of aCD3 and aCD28 (2.5 ng/mL and 100 ng/mL respectively) in a final assay volume of 200 mL. Supernatants were collected after 48 hours, and analyzed using the Meso Scale Discovery (MSD) SECTOR™ Imager 6000 (Meso Scale Discovery, Gaithersbury, Md.) to measure the IL-2 and IFNg protein levels by utilizing electrochemiluminescence (ECL). 20 mL of supernatant was added to the MSD multi-spot 96-well plates (each well containing IL-2, TNFa, and IFNg capture antibodies). The plates were sealed and incubated at room temperature on a plate shaker for 1 hour. Then 20 mL of Ruthenium-labeled detection antibodies (1 μg/ml final of IL-2, TNFα, and IFNγ in Antibody Dilution Buffer) and 110 mL of 2×MSD Read Buffer were added. The plates were sealed, covered with tin foil, and incubated at room temperature on a plate shaker for 1 hour. The plates were then read on the SECTOR™ Imager 6000. FIGS. 37A & 37B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg production from T cells in a dose-dependent manner. Compared to native ShK[1-35] peptide which shows only partial inhibition, the peptibody produces nearly complete inhibition of the inflammatory cytokine response. (POC=Percent Of Control of response in the absence of inhibitor).

Inhibition of T cell proliferation following anti-CD3 and anti-CD28 antibody stimulation of PBMCs. PBMC's were previously isolated from normal human donor Leukophoresis packs, purified by density gradient centrifugation (Ficoll Hypaque), cryopreserved in CPZ Cryopreservation Medium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's were thawed (95% viability), washed, and seeded at 2×10⁵ cells per well in culture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 100 μM non-essential amino acids, and 20 μM 2-ME) in 96-well flat-bottom tissue culture plates. Cells were pre-incubated with either anti-human CD32 (FcyRII) blocking antibody (per manufacturers instructions EASY SEP Human Biotin Selection Kit #18553, StemCell Technologies Vancouver, BC) or Fc-L10-ShK (100 nM-0.001 nM final) for 45 min. Fc-L10-ShK (100 nM-0.001 nM final) was then added to the cells containing anti-human CD32 blocking antibody while medium was added to the cells containing Fc-L10-ShK. Both sets were incubated for an additional 45 min before stimulating for 48 hr with aCD3/aCD28 (0.2 ng/ml and 100 ng/ml, respectively). Final assay volume was 200 ul. [3H]TdR (1 uCi per well) was added and the plates were incubated for an additional 16 hrs. Cells were then harvested onto glass fiber filters and radioactivity was measured in a B-scintillation counter. FIGS. 36A & 36B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits proliferation of T cells in a dose-dependent manner. Pre-blocking with the anti-CD32 (FcR) blocking antibody has little effect on the peptibodies ability to inhibit T cell proliferation suggesting Kv1.3 inhibition and not FcR binding is the mechanism for the inhibition observed (POC=Percent Of Control of response in the absence of inhibitor).

Immunohistochemistry analysis of Fc-L10-ShK[1-35] binding to HEK 293 cells overexpressing human Kv1.3. HEK 293 cells overexpressing human Kv1.3 (HEK Kv1.3) were obtained from BioFocus plc (Cambridge, UK) and maintained per manufacturer's recommendation. The parental HEK 293 cell line was used as a control. Cells were plated on Poly-D-Lysine 24 well plates (#35-4414; Becton-Dickinson, Bedford, Mass.) and allowed to grow to approximately 70% confluence. HEK KV1.3 were plated at 0.5×10e5 cells/well in 1 ml/well of medium. HEK 293 cells were plated at a density of 1.5×10e5 cells/well in 1 ml/well of medium. Before staining, cells were fixed with formalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5% BSA before use) by removing cell growth medium, adding 0.2 ml/well formalin solution and incubating at room temperature for ten minutes. Cells were stained by incubating with 0.2 ml/well of 5 μg/ml Fc-L10-ShK[1-35] in PBS/BSA for 30′ at room temperature. Fc-L10-ShK[1-35] was aspirated and then the cells were washed one time with PBS/0.5% BSA. Detection antibody (Goat F(ab)2 anti-human IgG-phycoerythrin; Southern Biotech Associates, Birmingham, Ala.) was added to the wells at 5 μg/ml in PBS/0.5% BSA and incubated for 30′ at room temperature. Wash cells once with PBS/0.5% BSA and examine using confocal microscopy (LSM 510 Meta Confocal Microscope; Carl Zeiss AG, Germany). FIG. 33B shows the Fc-L10-ShK[1-35] peptibody retains binds to Kv1.3 overexpressing HEK 293 cells but shows little binding to untransfected cells (FIG. 33A) indicating the Fc-L10-ShK[1-35] peptibody can be used as a reagent to detect cells overexpressing the Kv1.3 channel. In disease settings where activated T effector memory cells have been reported to overproduce Kv1.3, this reagent can find utility in both targeting these cells and in their detection.

An ELISA assay demonstrating Fc-L10-ShK[1-35] binding to fixed HEK 293 cells overexpressing Kv1.3. FIG. 34A shows a dose-dependent increase in the peptibody binding to fixed cells that overexpress Kv1.3, demonstrating that the peptibody shows high affinity binding to its target and the utility of the Fc-L10-ShK[1-35] molecule in detection of cells expressing the channel. Antigen specific T cells that cause disease in patients with multiple sclerosis have been shown to overexpress Kv1.3 by whole cell patch clamp electrophysiology,—a laborius approach. Our peptibody reagent can be a useful and convenient tool for monitoring Kv1.3 channel expression in patients and have utility in diagnostic applications. The procedure shown in FIG. 34A and FIG. 34B follows.

FIG. 34A. A whole cell immunoassay was performed to show binding of intact Fc-L10-ShK[1-35] to Kv1.3 transfected HEK 293 cells (BioFocus plc, Cambridge, UK). Parent HEK 293 cells or HEK Kv1.3 cells were plated at 3×10e4 cells/well in poly-D-Lysine coated ninety-six well plates (#35-4461; Becton-Dickinson, Bedford, Mass.). Cells were fixed with formalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5% BSA before use) by removing cell growth medium, adding 0.2 ml/well formalin solution and incubating at room temperature for 25 minutes and then washing one time with 100 μl/well of PBS/0.5% BSA. Wells were blocked by addition of 0.3 ml/well of BSA blocker (50-61-00; KPL 10% BSA Diluent/Blocking Solution, diluted 1:1 with PBS; KPL, Gaithersburg, Md.) followed by incubation at room temperature, with shaking, for 3 hr. Plates were washed 2 times with 1×KP Wash Buffer (50-63-00; KPL). Samples were diluted in Dilution Buffer (PBS/0.5% Tween-20) or Dilution Buffer with 1% Male Lewis Rat Serum (RATSRM-M; Bioreclamation Inc., Hicksville, N.Y.) and 0.1 ml/well was added to blocked plates, incubating for 1 hr at room temperature with shaking. Plates were washed 3 times with 1×KP Wash Buffer and then incubated with HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford, Ill.) diluted 1:5000 in PBS/0.1% Tween-20 for 1 hr at room temperature, with shaking. Plates were washed plates 3 times with 1×KP Wash Buffer, and then 0.1 ml/well TMB substrate (52-00-01; KPL) was added. The reactions were stopped by addition of 0.1 ml/well 2 N Sulfuric Acid. Absorbance was read at 450 nm on a Molecular Devices SpectroMax 340 (Sunnyvale, Calif.).

FIG. 34B. Whole cell immunoassay was performed as above with the following modifications. HEK 293 cells were plated at 1×10e5 cells/well and HEK Kv1.3 cells were plated at 6×10e4 cells/well in poly-D-Lysine coated 96 well plates. Fc Control was added at 500 ng/ml in a volume of 0.05 ml/well. HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford, Ill.) was diluted 1:10,000 in PBS/0.1% Tween-20. ABTS (50-66-00, KPL) was used as the substrate. Absorbances were read at 405 nm after stopping reactions by addition of 0.1 ml/well of 1% SDS.

Example 38 Purification of Fc-L10-ShK(1-35)

Expression of Fc-L10-ShK[1-35] was as described in Example 3 herein above. Frozen, E. coli paste (18 g) was combined with 200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet (3.2 g) was then dissolved in 32 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution was then centrifuged at 27,000 g for 15 min at room temperature, and then 5 ml of the supernatant was transferred to 500 ml of the refolding buffer (3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then stored at −70° C.

The stored refold was defrosted and then diluted with 2 L of water and the pH was adjusted to 7.3 using 1 M H₃PO₄. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 10 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 1 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125 volumes (25 ml) of 3 M sodium acetate was added.

A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 46A). The concentration of the filtered material was determined to be 2.56 mg/ml using a calculated molecular mass of 30,410 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 46B). The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 46C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. The product was then stored at −80° C.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-ShK[1-35], also referred to as “Fc-L-ShK[1-35]”, is shown in Table 35 (in Example 50 herein below).

Example 39 Purification of Bacterially Expressed Fc-L10-ShK(2-35)

Expression of Fc-L10-ShK[2-35] was as described in Example 4 herein above. Frozen, E. coli paste (16.5 g) was combined with 200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet (3.9 g) was then dissolved in 39 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution was then centrifuged at 27,000 g for 15 min at room temperature, and then 5 ml of the supernatant was transferred to 500 ml of the refolding buffer (3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then stored at −70° C.

The stored refold was defrosted and then diluted with 2 L of water and the pH was adjusted to 7.3 using 1 M H₃PO₄. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-Buffer A (20 mM NaH₂PO₄, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 10 ml/min 7° C. The fractions containing the desired product were pooled and filtered through a 0.22 μm cellulose acetate filter. The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH₂PO₄ pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125 volumes (18 ml) of 3 M sodium acetate was added, and the sample was filtered through a 0.22 μm cellulose acetate filter.

A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 40A). The concentration of the filtered material was determined to be 3.20 mg/ml using a calculated molecular mass of 29,282 g/mol and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 40B). The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 40C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 40D). The product was then stored at −80° C.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-ShK[2-35], also referred to as “Fc-L-ShK[2-35]”, is shown in Table 35 (in Example 50 herein below).

Example 40 Purification of Bacterially Expressed Fc-L10-OsK1

Frozen, E. coli paste (129 g; see Example 10) was combined with 1290 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.8 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 15 min at 4° C. The pellet was then resuspended in 1290 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 15 min at 4° C. The pellet was then resuspended in 1290 ml water using a tissue grinder and then centrifuged at 17,700 g for 15 min at 4° C. 8 g of the pellet (16.3 g total) was then dissolved in 160 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 100 ml of the pellet solution was then incubated with 1 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 5000 ml of the refolding buffer (1 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 10.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.

The pH of the refold was adjusted to 8.0 using acetic acid. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 50 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 50 Amersham Protein A column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the protein A column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product were pooled and immediately loaded on to a 50 ml Amersham SP-Sepharose HP column (2.6 cm I.D.) at 20 ml/min in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled and then applied to a 75 ml MEP Hypercel column (2.6 cm I.D.) at 5 ml/min in MEP Buffer A (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with a linear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH 4.0) followed by a step to 100% MEP Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the bulk of the desired product were pooled.

The MEP pool was then concentrated to about 20 ml using a Pall Jumbo-Sep with a 10 kDa membrane followed by buffer exchange with Formulation Buffer (20 mM NaH₂PO₄, 200 mM NaCl, pH 7.0) using the same membrane. A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl Formulation Buffer using a Hewlett Packard 8453 spectrophotometer (FIG. 41A). The concentration of the material was determined to be 4.12 mg/ml using a calculated molecular mass of 30,558 g/mol and extinction coefficient of 35,720 M⁻¹ cm⁻¹. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 41B). The macromolecular state of the product was then determined using size exclusion chromatography on 123 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 41C). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 41D). The product was filtered through a 0.22 μm cellulose acetate filter and then stored at −80° C.

The yield for the E. coli-expressed Fc-L10-OSK1 prep was 81 mg from 40 g of cell paste (129 g×(8 g/16.3 g)×(100 ml/160 ml)=39.6 g which was rounded to 40 g), the purity was greater than 80% judging by SDS-PAGE, it is running as the expected dimer judging by SEC-HPLC, and the mass was within the expected molecular weight range judging by MS.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-OSK1, also referred to as “Fc-L-OSK1”, is shown in Table 35 (in Example 50 herein below).

Example 41 Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D] Expressed by Mammalian Cells

Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D], inhibitors of Kv1.3, were expressed in mammalian cells. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a linker sequence and a monomer of the Kv1.3 inhibitor peptide OSK1, OSK1[K7S], OSK1[E16K,K20D], or OSK1[K7S,E16K,K20D] was constructed as described below. Methods for expressing and purifying the peptibody from mammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosed herein.

For construction of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] expression vectors, a PCR strategy was employed to generate the full length genes, OSK1, OSK1[K7S], OSK1[E16K,K20D], and OSK1[K7S,E16K,K20D], each linked to a four glycine and one serine amino acid linker with two stop codons and flanked by BamHI and NotI restriction sites as shown below.

Two oligos for each of OSK1, OSK1[K7S], OSK1[E16K,K20D], and [K7S,E16K,K20D]OSK1 with the sequence as depicted below were used in a PCR reaction with PfuTurbo HotStart DNA polymerase (Stratagene) at 95° C.-30 sec, 55° C.-30 sec, 75° C.-45 sec for 35 cycles; BamHI (ggatcc) and NotI (gcggccgc) restriction sites are underlined.

OSK1: Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 876) ggc gtg atc atc aac gtg aag tgc aag atc agc cgc cag tgc ctg gag ccc tgc aag aag gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg gtg (SEQ ID NO: 877) cag tgg cac ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcc ttc ttg cag ggc tcc a; OSK1[K7S]: Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 878) ggc gtg atc atc aac gtg agc tgc aag atc agc cgc cag tgc ctg gag ccc tgc aag aag gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg gtg (SEQ ID NO: 879) cag tgg cac ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcc ttc ttg cag ggc tcc a; OSK1[E16K, K20D]: Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 880) ggc gtg atc atc aac gtg aag tgc aag atc agc cgc cag tgc ctg aag ccc tgc aag gac gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg gtg (SEQ ID NO: 881) cag tgg cac ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcg tcc ttg cag ggc ttc a; OSK1[K7S, E16K, K20D]: Forward primer: cat gga tcc gga gga gga gga agc (SEQ ID NO: 882) ggc gtg atc atc aac gtg agc tgc aag atc agc cgc cag tgc ctg aag ccc tgc aag gac gcc g; Reverse primer: cat gcg gcc gct tac tac ttg ggg gtg (SEQ ID NO: 883) cag tgg cac ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcg tcc ttg cag ggc ttc a.

The resulting PCR products were resolved as the 155 bp bands on a four percent agarose gel. The 155 bp PCR product was purified using PCR Purification Kit (Qiagen), then digested with BamHI and NotI (Roche) restriction enzymes, and agarose gel was purified by Gel Extraction Kit (Qiagen). At the same time, the pcDNA3.1(+) CMVi-hFc-Shk[2-35] vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Extraction Kit. The gel purified PCR fragment was ligated to the purified large fragment and transformed into One Shot® Top10F′ (Invitrogen). DNAs from transformed bacterial colonies were isolated and digested with BamHI and NotI restriction enzymes and resolved on a two percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequences, only one clone from each gene was selected for large scaled plasmid purification. The DNA of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi vector was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the above sequences. The sequences and pictorial representations of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] are depicted in FIG. 42A-B, FIG. 43A-B, FIG. 44A-B and FIG. 45A-B, respectively.

HEK-293 cells used in transient transfection expression of Fc-L110-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi protein were cultured in growth medium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS from Gibco), 1× non-essential amino acid (NEAA from Gibco) and 1× Penicillin/Streptomycine/Glutamine (Pen/Strep/Glu from Gibco). 5.6 μg each of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi plasmid that had been phenol/chloroform extracted was transfected into HEK-293 cells using FuGENE 6 (Roche). The cells were recovered for 24 hours, and then placed in DMEM High Glucose, 1×NEAA and 1× Pen/Strep/Glu medium for 48 hours. Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] were purified from medium conditioned by these transfected HEK-293 cells using a protocol described in Example 50 herein below.

Fifteen μl of conditioned medium was mixed with an in-house 4× Loading Buffer (without β-mercaptoethanol) and electrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2 hours in a 1× Gel Running solution (25 mM Tris Base, 192 mM Glycine, 3.5 mM SDS) along with 20 μl of BenchMark Pre-Stained Protein ladder (Invitrogen). The gel was then soaked in Electroblot buffer (25 mM Tris base, 192 mM glycine, 20% methanol,) for 5 minutes. -A nitrocellulose membrane from Invitrogen (Cat. No. LC200, 0.2 μm pores size) was soaked in Electroblot buffer. The pre-soaked gel was blotted to the nitrocellulose membrane using the Mini Trans-Blot Cell module according to the manufacturer instructions (Bio-Rad Laboratories) at 300 mA for 2 hours. The blot was rinsed in Tris buffered saline solution pH7.5 with 0.1% Tween20 (TBST). Then, the blot was first soaked in a 5% milk (Camation) in TBST for 1 hour at room temperature, followed by washing three times in TBST for 10 minutes per wash. Then, incubated with 1:1000 dilution of the HRP-conjugated Goat anti-human IgG, (Fcγ) antibody (Pierece Biotechnology Cat. no. 31413) in TBST with 5% milk buffer for 1 hour with shaking at room temperature. The blot was then washed three times in TBST for 15 minutes per wash at room temperature. The primary antibody was detected using Amersham Pharmacia Biotech's ECL western blotting detection reagents according to manufacturers instructions. Upon ECL detection, the western blot analysis displayed the expected size of 66 kDa under non-reducing gel conditions (FIG. 46).

Plasmids containing the Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] inserts in pCMVi vector were digested with XbaI and NotI (Roche) restriction enzymes and gel purified. The inserts were individually ligated into SpeI and NotI (Roche) digested pDSRα24 (Amgen Proprietary) expression vector. Integrity of the resulting constructs were confirmed by DNA sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification.

AM1 CHOd- (Amgen Proprietary) cells used in the stable expression of Fc-L10-OSK1 protein were cultured in AM1 CHOd- growth medium containing DMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HT from Gibco), 1×NEAA and 1× Pen/Strep/Glu. 5.6 μg of pDSRα-24-Fc-L10-OSK1 plasmid was transfected into AM1 CHOd- cells using FuGene 6. Twenty-four hours post transfection, the cells were split 1:11 into DHFR selection medium (DMEM High Glucose plus 10% Dialyzed Fetal Bovine Serum (dFBS), 1×NEAA and 1× Pen/Strep/Glu) at 1:50 dilution for colony selection. The cells were selected in DHFR selection medium for thirteen days. The ten 10-cm² pools of the resulting colonies were expanded to ten T-175 flasks, then were scaled up ten roller bottles and cultured under AM1 CHOd- production medium (DMEM/F12 (1:1), 1×NEAA, 1× Sodium Pyruvate (Na Pyruvate), 1× Pen/Strep/Glu and 1.5% DMSO). The conditioned medium was harvested and replaced at one-week intervals. The resulting six liters of conditioned medium were filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysis as shown in FIG. 47. Then, transferred to Protein Chemistry for purification.

Twelve colonies were selected after 13 days on DHFR selection medium and picked into one 24-well plate. The plate was allowed to grow up for one week, and then was transferred to AM1 CHOd- production medium for 48-72 hours and the conditioned medium was harvested. The expression levels were evaluated by Western blotting similar to the transient Western blot analysis with detection by the same HRP-conjugated Goat anti-human IgG, (Fcγ) antibody to screen 5 μl of conditioned medium. All 12 stable clones exhibited expression at the expected size of 66 kDa. Two clones, A3 and C2 were selected and expanded to T175 flask for freezing with A3 as a backup to the primary clone C2 (FIG. 48).

The C2 clone was scaled up into fifty roller bottles (Corning) using selection medium and grown to confluency. Then, the medium was exchanged with a production medium, and let incubate for one week. The conditioned medium was harvested and replaced at the one-week interval. The resulting fifty liters of conditioned medium were filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysis (data not shown). Further purification was accomplished as described in Example 42 herein below.

Example 42 Purification of Fc-L10-OSK1, Fc-L10-OSK1(K7S), Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D) Expressed by Mammalian Cells

Purification of Fc-L10-OSK1. Approximately 6 L of CHO (AM1 CHOd-) cell-conditioned medium (see, Example 41 above) was loaded on to a 35 ml MAb Select column (GE Healthcare) at 10 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The MAb Select elution was directly loaded on to an inline 65 ml SP-HP column (GE Healthcare) in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 10 ml/min 7° C. After disconnecting the MAb select column, the SP-HP column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 5% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) at 10 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 20 ml using a Pall Life Sciences Jumbosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then buffer exchanged by diluting with 20 ml of 20 mM NaH₂PO₄, pH 7.0 and reconcentrated to 20 ml using the Jumbosep 10K Omega filter. The material was then diluted with 20 ml 20 mM NaH₂PO₄, 200 mM NaCl, pH 7.0 and then reconcentrated to 22 ml. The buffer exchanged material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 1 ml/min room temperature. A spectral scan was then conducted on 50 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 49A, black trace). The concentration of the filtered material was determined to be 4.96 mg/ml using a calculated molecular mass of 30,371 g/mol and extinction coefficient of 35,410 M⁻¹ cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 49B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 30-fold dilution of the sample in Charles Rivers Laboratories Endotoxin Specific Buffer yielding a result of 1.8 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 149 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 49C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. (FIG. 49D). The product was then stored at −80° C.

The yield for the mammalian Fc-L10-OSK1 prep was 115 mg from 6 L, the purity was >90% judging by SDS-PAGE; Fc-L10-OSK1 ran as the expected dimer judging by SEC-HPLC, and the mass is with the expected range judging by MS.

The activity of purified Fc-L10-OSK1 in blocking human Kv1.3 and human Kv1.1 is described in Example 43 herein below.

Purification of Fc-L10-OSK1(K7S). Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D). Approximately 500 mL of medium conditioned by transfected HEK-293 (see, Example 41 above) was combined with a 65% slurry of MAb Select resin (1.5 ml) (GE Healthcare) and 500 μl 20% NaN₃. The slurry was then gently agitated for 3 days at 4° C. followed by centrifugation at 1000 g for 5 minutes at 4° C. using no brake. The majority of the supernatant was then aspirated and the remaining slurry in the pellet was transferred to a 14 ml conical tube and combined with 12 ml of Dulbecco's phosphate buffered saline without divalent cations (PBS). The slurry was centrifuged at 2000 g for 1 minute at 4° C. using a low brake and the supernatant was aspirated. The PBS wash cycle was repeated an additional 3 times. The bound protein was then eluted by adding 1 ml of 100 mM glycine pH 3.0 and gently agitating for 5 min at room temperature. The slurry was then centrifuged at 2000 g for 1 minute at 4° C. using a low brake and the supernatant was aspirated as the first elution. The elution cycle was repeated 2 more times, and all 3 supernatants were combined into a single pool. Sodium acetate (37.5 μl of a 3 M solution) was added to the elution pool to raise the pH, which was then dialyzed against 10 mM acetic acid, 5% sorbitol, pH 5.0 for 2 hours at room temperature using a 10 kDa SlideAlyzer (Pierce). The dialysis buffer was changed, and the dialysis continued over night at 4° C. The dialyzed material was then filtered through a 0.22 μm cellulose acetate filter syringe filter. Then concentration of the filtered material was determined to be 1.27 mg/ml using a calculated molecular mass of 30,330 and extinction coefficient of 35,410 M⁻¹ cm⁻¹ (FIG. 50A). The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 50B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 25-fold dilution of the sample in Charles Rivers Laboratories Endotoxin Specific Buffer yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 50C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. (FIG. 50D). The product was then stored at −80° C.

FIGS. 51A-D show results from the purification and analysis for Fc-L10-OsK1(E16K, K20D), which was conducted using the same protocol as that for the Fc-L110-OsK1 (K7S) molecule (described above) with the following exceptions: the concentration was found to be 1.59 mg/ml using a calculated molecular mass of 30,357 g/mol and a calculated extinction coefficient of 35,410; the pyrogen level was found to be <1 EU/mg using a 32-fold dilution.

FIGS. 52A-D show results from the purification and analysis for Fc-L10-OsK1(K7S,E16K, K20D), which was conducted using the same protocol as that for the Fc-L10-OsK1(K7S) molecule (described above) with the following exceptions: the concentration was found to be 0.81 mg/ml using a calculated molecular mass of 30,316 g/mol and a calculated extinction coefficient of 35,410; the pyrogen level was found to be <1 EU/mg using a 16-fold dilution.

The activity of purified Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K, K20D] and Fc-L10-OSK1[K7S, E16K, K20D] in blocking human Kv1.3 and human Kv1.1 is described in Example 43 herein below.

Example 43 Electrophysiology of OSK1 and OSK1 Peptibody Analogs

A 38-residue peptide toxin of the Asian scorpion Orthochirus scrobiculosus venom (OSK1) was synthesized (see, Examples 41) to evaluate its impact on the human Kv1.1 and Kv1.3 channels, subtypes of the potassium channel family. The potency and selectivity of synthetic OSK1 in inhibiting the human Kv1.1 and Kv1.3 channels was evaluated by the use of HEK293 cell expression system and electrophysiology (FIG. 53). Whole cell patch clamp recording of stably expressed Kv1.3 channels revealed that the synthetic OSK1 peptide is more potent in inhibiting human Kv1.3 when compared to Kv1.1 (Table 33).

Fusion of OSK1 peptide toxin to antibody to generate OSK1 peptibody. To improve plasma half-life and prevent OSK1 peptide toxin from penetrating the CNS, the OSK1 peptide toxin was fused to the Fc-fragment of a human antibody IgG1 via a linker chain length of 10 amino acid residues (Fc-L10-OSK1), as described in Example 41 herein. This fusion resulted in a decrease in the potency of Kv1.3 by 5-fold when compared to the synthetic OSK1 peptide. However, it significantly improved the selectivity of OSK1 against Kv1.1 by 210-fold when compared to that of the synthetic peptide alone (4-fold; Table 33 and FIG. 54).

Modification of OSK1-peptibody (Fc-L10-OSK1). OSK1 shares 60 to 80% sequence homology to other members of scorpion toxins, which are collectively termed α-KTx3. Sequence alignment of OSK1 and other members of α-KTx3 family revealed 4 distinct structural differences at positions 12, 16, 20, and 36. These structural differences of OSK1 have been postulated to play an important role in its wide range of activities against other potassium channels, which is not observed with other members of α-KTx3 family. Hence, two amino acid residues at position 16 and 20 were restored to the more conserved amino acid residues within the OSK1 sequence in order to evaluate their impact on selectivity against other potassium channels such as Kv1.1, which is predominantly found in the CNS as a heterotetromer with Kv1.2. By substituting for glutamic acid at position 16, and for lysine at position 20, the conserved lysine and aspartic acid residues, respectively (i.e., Fc-L10-OSK1[E16K, K20D]), we did not observe a significant change in potency when compared to that of Fc-L10-OSK1 (1.3-fold difference; FIG. 56 and Table 33). However, this double mutation removed the blocking activity against Kv1.1. The selectivity ratio of Kv1.1/Kv1.3 was 403-fold, which was a significant improvement over the selectivity ratio for Fc-L10-OSK1 (210-fold). A single amino acid mutation at position 7 from lysine to serine (Fc-L10-OSK1[K7S]) produced a slight change in potency and selectivity by 2- and 1.3-fold, respectively, when compared to those of Fc-L10-OSK1 (FIG. 55 and Table 33). There was a significant decrease in potency as well as selectivity when all three residues were mutated to generate Fc-L10-OSK1[K7S, E16K, K20D] (FIG. 57 and Table 33).

As demonstrated by the results in Table 33, we dramatically improved selectivity against Kv1.1 by fusing the OSK1 peptide toxin to the Fc-fragment of the human antibody IgG1, but reduced target potency against Kv1.3. The selectivity against Kv1.1 was further improved when 2 residues at two key positions were restored to the conserved residues found in other members of the α-KTx3 family.

Table 33shows a summary of IC50 values for OSK1 analogs against hKv1.3 and hKv1.1 channels. All analogues are ranked based on their potency against hKv1.3. Also shown in the table is the selectivity ratio of hKv1.1/hKv1.3 for all OSK1 analogs.

hKv1.3: IC₅₀ hKv1.1: IC₅₀ hKv1.1/ Compound [pM] [pM] hKv1.3 Synthetic OSK1 39 160 4 Fc-L10-OSK1 198 41600 210 Fc-L10-OSK1[E16K, K20D] 248 100000 403 Fc-L10-OSK1[K7S] 372 100000 269 Fc-L10-OSK1[K7S, E16K, K20D] 812 10000 12

Example 44 Pharmacokinetic Study of PEG-ShK[1-35] Molecule in Rats

The intravenous (IV) pharmacokinetic profile was determined of a about 24-kDa 20K PEG-ShK[1-35] molecule and the about 4-kDa small native ShK peptide was determined in Spraque Dawley rats. The IV dose for the native ShK peptide and our novel 20K PEG-ShK[1-35] molecule was 1 mg/kg. This dose represented equal molar amounts of these two molecules. The average weight of the rats was about 0.3 kg and two rats were used for each dose & molecule. At various times following IV injection, blood was drawn and about 0.1 ml of serum was collected. Serum samples were stored frozen at −80° C. until analysis.

Assay Plate preparation for electrophysiology. Rat serum samples containing the 20K PEG-ShK[1-35] molecule or the native ShK peptide from pharmacokinetic studies were received frozen. Before experiments, each sample was thawed at room temperature and an aliquot (70 to 80 μl) was transferred to a well in a 96-well polypropylene plate. In order to prepare the Assay Plate, several dilutions were made from the pharmacokinetic serum samples to give rise to Test Solutions. Dilutions of serum samples from the pharmacokinetic study were into 10% Phosphate Buffered Saline (PBS, with Ca²⁺ and Mg²⁺). For determination of the amount of our novel 20K PEG-ShK[1-35] molecule in serum samples from the pharmacokinetic study, the final serum concentrations in the Test Solutions were 90%, 30%, 10%, 3.3% and 1.1%. Purified 20K PEG-Shk[1-35] Standard inhibition curves were also prepared in the Assay Plate. To do this, 8-point serial dilutions of the purified 20K PEG-ShK[1-35] molecule (Standard) were prepared in either 90%, 30%, 10%, 3.3% or 1.1% rat serum and the final concentration of standard was 50, 16.7, 5.5, 1.85, 0.62, 0.21, 0.068 and 0.023 nM.

Cell preparation for electrophysiology. CHO cells stably expressing the voltage-activated K⁺ channel, K_(V)1.3 were plated in T-175 tissue culture flasks (at a density of 5×10⁶) 2 days before experimentation and allowed to grow to around 95% confluence. Immediately prior to the experiment, the cells were washed with PBS and then detached with a 2 ml mixture (1:1 volume ratio) of trypsin (0.25%) and versene (1:5000) at 37° C. (for 3 minutes). Subsequently, the cells were re-suspended in the flask in 10 ml of tissue culture medium (HAM's F-12 with Glutamax, Invitrogen, Cat#31765) with 10% FBS, 1×NEAA and 750 μg/ml of G418) and centrifuged at about 1000 rpm for 1½ minutes. The resultant cell pellet was re-suspended in PBS at 3-5×10⁶ cells/ml.

IonWorks electrophysiology and data analysis. The ability of Test solutions or Standards in serum to inhibit K⁺ currents in the CHO-K_(v)1.3 cells was investigated using the automated electrophysiology system, IonWorks Quattro. Re-suspended cells, the Assay Plate, a Population Patch Clamp (PPC) PatchPlate as well as appropriate intracellular (90 mMK-Gluconate, 20 mMKF, 2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35) and extracellular (PBS, with Ca²⁺ and Mg²⁺) buffers were positioned on IonWorks Quattro. Electrophysiology recordings were made from the CHO-K_(v)1.3 cells using an amphotericin-based perforated patch-clamp method. Using the voltage-clamp circuitry of the IonWorks Quattro, cells were held at a membrane potential of −80 mV and voltage-activated K⁺ currents were evoked by stepping the membrane potential to +30 mV for 400 ms. K⁺ currents were evoked under control conditions i.e., in the absence of inhibitor at the beginning of the experiment and after 10-minute incubation in the presence of the Test Solution or Standard. The mean K⁺ current amplitude was measured between 430 and 440 ms and the data were exported to a Microsoft Excel spreadsheet. The amplitude of the K⁺ current in the presence of each concentration of the Test Solution or Standard was expressed as a percentage of the K⁺ current in control conditions in the same well.

Standard inhibition curves were generated for each standard in various levels of rat serum and expressed as current percent of control (POC) versus log of nM concentration. Percent of control (POC) is inversely related to inhibition, where 100 POC is no inhibition and 0 POC is 100% inhibition. Linear regression over a selected region of the curve was used to derive an equation to enable calculation of drug concentrations within Test solutions. Only current values within the linear portion of the Standard curve were used to calculate the concentration of drug in Test solutions. The corresponding Standard curve in a given level of serum, was always compared to the same level of serum of Test solution when calculating drug level. The Standard curves for ShK and 20K PEG-ShK[1-35] are shown in FIG. 58A and FIG. 58B, respectively, and each figure contains linear regression equations for each Standard at a given percentage of serum. For the 20K PEG-ShK[1-35] standard curve the linear portion of the Standard curve was from 20 POC to 70 POC and only current values derived from the Test solution which fell within this range were used to calculate drug concentration within the Test solution.

The pharmacokinetic profile of our novel 20K PEG ShK[1-35] molecule after IV injection is shown in FIG. 59. The terminal half-life (t_(1/2) b) of this molecule is estimated from this curve to be between 6 to 12 hours long. Beyond 48 hours, the level of drug falls outside the linear range of the Standard curve and is not calculated. The calculated 6 to 12 hour half-life of our novel 20K PEG-ShK[1-35] molecule was substantially longer than the approximately 0.33 hour (or 20 min) half-life of the native ShK molecule reported earlier by C. Beeton et al. [C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942-13947], and is a desirable feature of a therapeutic molecule. A comparison of the relative levels of Kv1.3 inhibitor after an equal molar IV injection of ShK versus 20K PEG-ShK[1-35] is shown in FIG. 60. As can be seen from this figure examining 5% serum Test solutions, the 20K PEG-ShK[1-35] molecule showed significant suppression of Kv1.3 current (<70 POC) for more than 24 hours, whereas the native ShK peptide only showed a significant level of inhibition of Kv1.3 current for the first hour and beyond 1 hour showed no significant blockade. These data again demonstrate a desirable feature of the 20K PEG ShK[1-35] molecule as a therapeutic for treatment of autoimmune disease.

Example 45 PEGylated Toxin Peptide Suppressed Severe Autoimmune Encephalomyelitis in Animal Model

The 20KPEG-ShK inhibitor of Kv1.3 shows improved efficacy in suppressing severe autoimmune encephalomyelitis in rats. Using an adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis described earlier [C. Beeton et al. (2001) J. Immunol. 166, 936], we examined the activity in vivo of our novel 20KPEG-ShK molecule and compared its efficacy to that of the ShK toxin peptide alone. The study design is illustrated in FIG. 61. The results from this in vivo study are provided in FIG. 62 and FIG. 63. The 20KPEG-ShK molecule delivered subcutaneously (SC) at 10 μg/kg daily from day −1 to day 3 significantly reduced disease severity and increased survival, whereas animals treated with an equal molar dose (10 μg/kg) of the small ShK peptide developed severe disease and died.

The 35-amino acid toxin peptide ShK (Stichodactyla helianthus neurotoxin) was purchased from Bachem Bioscience Inc and confirmed by electrophysiology to potently block Kv1.3 (see Example 36 herein). The synthesis, PEGylation and purification of the 20KPEG ShK molecule was as described herein above. The encephalomyelogenic CD4+ rat T cell line, PAS, specific for myelin-basic protein (MBP) originated from Dr. Evelyne Beraud. The maintenance of these cells in vitro and their use in the AT-EAE model has been described earlier [C. Beeton et al. (2001) PNAS 98, 13942]. PAS T cells were maintained in vitro by alternating rounds of antigen stimulation or activation with MBP and irradiated thymocytes (2 days), and propagation with T cell growth factors (5 days). Activation of PAS T cells (3×10⁵/ml) involved incubating the cells for 2 days with 10 μg/ml MBP and 15×10⁶/ml syngeneic irradiated (3500 rad) thymocytes. On day 2 after in vitro activation, 10-15×10⁶ viable PAS T cells were injected into 6-12 week old female Lewis rats (Charles River Laboratories) by tail IV. Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS), 20KPEG-ShK or ShK were given from days −1 to 3 (FIG. 61), where day −1 represent 1 day prior to injection of PAS T cells (day 0). In vehicle treated rats, acute EAE developed 4 to 5 days after injection of PAS T cells (FIG. 62). Serum was collected by retro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end of the study) for analysis of levels of inhibitor. Rats were weighed on days −1, 4, 6, and 8. Animals were scored blinded once a day from the day of cell transfer (day 0) to day 3, and twice a day from day 4 to day 8. Clinical signs were evaluated as the total score of the degree of paresis of each limb and tail. Clinical scoring: 0=No signs, 0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hind leg paralysis, 5.0=complete hind leg paralysis and incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of 5.5 were euthanized.

Treatment of rats with the Kv1.3 blocker PEG-ShK prior to the onset of EAE caused a lag in the onset of disease, inhibited the progression of disease, and prevented death in a dose-dependent manner (FIG. 62). Onset of disease in rats that were treated with the vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK was observed on day 4, compared to day 4.5 in rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK. In addition, rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all developed severe disease by the end of the study with an EAE score of 5.5 or above. In contrast, rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK, reached a peak clinical severity score average of <2, and all but one rat survived to the end of the study. Furthermore, we found that rat body weight correlated with disease severity (FIG. 63). Rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all lost an average of 31 g, 30 g, and 30 g, respectively, while rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK lost 18 g and 11 g, respectively. Rats in the latter two groups also appeared to be gaining weight by the end of the study, a sign of recovery. It should be noted that rats treated with 10 μg/kg ShK and 10 μg/kg PEG-ShK received molar equivalents of the ShK peptide. The significantly greater efficacy of the PEG-ShK molecule relative to unconjugated ShK, is likely due to the PEG-ShK molecule's greater stability and prolonged half-life in vivo (see, Example 44).

Example 46 Compositions Including Kv1.3 Antagonist Peptides Block Inflammation in Human Whole Blood

Ex vivo assay to examine impact of Kv1.3 inhibitors on secretion of IL-2 and IFN-g. Human whole blood was obtained from healthy, non-medicated donors in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derived Fc-L10-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as described herein at Example 4 and Example 39. The calcineurin inhibitor cyclosporin A was obtained from the Amgen sample bank, but is also available commercially from a variety of vendors. Ten 3-fold serial dilutions of inhibitors were prepared in DMEM complete media at 4× final concentration and 50 μl of each were added to wells of a 96-well Falcon 3075 flat-bottom microtiter plate. Whereas columns 1-5 and 7-11 of the microtiter plate contained inhibitors (each row with a separate inhibitor dilution series), 50 μl of DMEM complete media alone was added to the 8 wells in column 6 and 100 μl of DMEM complete media alone was added to the 8 wells in column 12. To initiate the experiment, 100 μl of whole blood was added to each well of the microtiter plate. The plate was then incubated at 37° C., 5% CO₂ for one hour. After one hour, the plate was removed and 50 μl of the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate, except the 8 wells in column 12. The plates were placed back at 37° C., 5% CO₂ for 48 hours. To determine the amount of IL-2 and IFN-g secreted in whole blood, 100 μl of the supernatant (conditioned media) from each well of the 96-well plate was transferred to a storage plate. For MSD electrochemilluminesence analysis of cytokine production, 20 μl of the supernatants (conditioned media) were added to MSD Multi-Spot Custom Coated plates (meso-scale.com). The working electrodes on these plates were coated with four Capture Antibodies (hIL-5, hIL-2, hIFNg and hIL-4) in advance. After addition of 20 μl of conditioned media to the MSD plate, 150 μl of a cocktail of Detection Antibodies and P4 Buffer were added to each well. The 150 μl cocktail contained 20 ul of four Detection Antibodies (hIL-5, hIL-2, hIFNg and hIL-4) at 1 μg/ml each and 130 ul of 2×P4 Buffer. The plates were covered and placed on a shaking platform overnight (in the dark). The next morning the plates were read on the MSD Sector Imager. Since the 8 wells in column 6 of each plate received only the thapsigargin stimulus and no inhibitor, the average MSD response here was used to calculate the “High” value for a plate. The calculate “Low” value for the plate was derived from the average MSD response from the 8 wells in column 12 which contained no thapsigargin stimulus and no inhibitor. Percent of control (POC) is a measure of the response relative to the unstimulated versus stimulated controls, where 100 POC is equivalent to the average response of thapsigargin stimulus alone or the “High” value. Therefore, 100 POC represents 0% inhibition of the response. In contrast, 0 POC represents 100% inhibition of the response and would be equivalent to the response where no stimulus is given or the “Low” value. To calculate percent of control (POC), the following formula is used: [(MSD response of well)−(“Low”)]/[(“High”)−(“Low”)]×100. The potency of the molecules in whole blood was calculated after curve fitting from the inhibition curve (IC) and IC50 was derived using standard curve fitting software. Although we describe here measurement of cytokine production using a high throughput MSD electrochemillumenescence assay, one of skill in the art can readily envision lower throughput ELISA assays are equally applicable for measuring cytokine production.

Ex vivo assay demonstrating Kv1.3 inhibitors block cell surface activation of CD40L & IL-2R. Human whole blood was obtained from healthy, non-medicated donors in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derived Fc-L110-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as described in Example 4 and Example 39. The calcineurin inhibitor cyclosporin A was obtained from the Amgen sample bank, but is also available commercially from a variety of vendors. The ion channel inhibitors ShK, IbTx or DTK-k were diluted into DMEM complete media to 4× of the final concentration desired (final=50 or 100 nM). The calcineurin inhibitor cyclosporin A was also diluted into DMEM complete media to 4× final concentration (final=10 μM). To appropriate wells of a 96-well Falcon 3075 flat-bottom microtiter plate, 50 μl of either DMEM complete media or the 4× inhibitor solutions were added. Then, 100 μl of human whole blood was added and the plate was incubated for 1 hour at 37° C., 5% CO₂. After one hour, the plate was removed and 50 μl of the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate containing inhibitor. To some wells containing no inhibitor but just DMEM complete media, thapsigargin was also added whereas others wells with just DMEM complete media had an additional 50 μl of DMEM complete media added. The wells with no inhibitor and no thapsigargin stimulus represented the untreated “Low” control. The wells with no inhibitor but which received thapsigargin stimulus represented the control for maximum stimulation or “High” control. Plates were placed back at 37° C., 5% CO₂ for 24 hours. After 24 hours, plates were removed and wells were process for FACS analysis. Cells were removed from the wells and washed in staining buffer (phosphate buffered saline containing 2% heat-inactivated fetal calf serum). Red blood cells were lysed using BD FACS Lysing Solution containing 1.5% formaldehyde (BD Biosciences) as directed by the manufacturer. Cells were distributed at a concentration of 1 million cells per 100 microliters of staining buffer per tube. Cells were first stained with 1 microliter of biotin-labeled anti-human CD4, washed, then stained simultaneously 1 microliter each of streptavidin-APC, FITC-labeled anti-human CD45RA, and phycoerythrin (PE)-labeled anti-human CD25 (IL-2Ra) or PE-labeled anti-human CD40L. Cells were washed with staining buffer between antibody addition steps. All antibodies were obtained from BD Biosciences (San Diego, Calif.). Twenty to fifty thousand live events were collected for each sample on a Becton Dickinson FACSCaliber (Mountain View, Calif.) flow cytometer and analyzed using FlowJo software (Tree Star Inc., San Carlos, Calif.). Dead cells, monocytes, and granulocytes were excluded from the analysis on the basis of forward and side scatter properties.

FIG. 64 and FIG. 67 demonstrate that Kv1.3 inhibitors ShK and Fc-L10-ShK[2-35] potently blocked IL-2 secretion in human whole blood, in addition to suppressing activation of the IL-2R on CD4+ T cells. The Kv1.3 inhibitor Fc-L10-ShK[2-35] was more than 200 times more potent in blocking IL-2 production in human whole blood than cyclosporine A (FIG. 64) as reflected by the IC50. FIG. 65 shows that Kv1.3 inhibitors also potently blocked secretion of IFNg in human whole blood, and FIG. 66 demonstrates that upregulation of CD40L on T cells was additionally blocked. The data in FIGS. 64-67 show that the Fc-L10-ShK[2-35] molecule was stable in whole blood at 37° C. for up to 48 hours, providing potent blockade of inflammatory responses. Toxin peptide therapeutic agents that target Kv1.3 and have prolonged half-life, are sought to provide sustained blockade of these responses in vivo over time. In contrast, despite the fact the Kv1.3 inhibitor peptide ShK also showed potent blockade in whole blood, the ShK peptide has a short (˜20 min) half-life in vivo (C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942), and cannot, therefore, provide prolonged blockade. Whole blood represents a physiologically relevant assay to predict the response in animals. The whole blood assays described here can also be used as a pharmacodynamic (PD) assay to measure target coverage and drug exposure following dosing of patients. These human whole blood data support the therapeutic usefulness of the compositions of the present invention for treatment of a variety immune disorders, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus.

Example 47 PEGylated Peptibodies

By way of example, PEGylated peptibodies of the present invention were made by the following method. CHO-expressed FcL10-OsK1 (19.2 mg; MW 30,371 Da, 0.63 micromole) in 19.2 ml A5S, 20 mM NaBH₃CN, pH 5, was treated with 38 mg PEG aldehyde (MW 20 kDa; 3×, Lot 104086). The sealed reaction mixture was stirred in a cold room overnight. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzed material was then loaded onto an SP HP FPLC column (16/10) in A5S pH 5 and eluted with a 1 M NaCl gradient. The collected fractions were analyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS, concentrated and submitted for functional testing (Table 34).

In another example, FcL10-ShK1 (16.5 mg; MW 30,065 Da, 0.55 micro mole) in 16.5 ml A5S, 20 mM NaBH₃CN, pH 5 was treated with 44 mg PEG aldehyde (MW 20 kDa; 4×, Lot 104086). The sealed reaction mixture was stirred in a cold room overnight. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzed material was loaded onto an SP HP FPLC column (16/10) in A5S pH 5 and was eluted with a 1 M NaCl gradient. The collected fractions were analyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS, concentrated and submitted for functional testing (Table 34).

The data in Table 34 demonstrate potency of the PEGylated peptibody molecules as Kv1.3 inhibitors.

Table 34 shows determinations of IC50 made by whole cell patch clamp electrophysiology with HEK 293 as described in Example 36 herein above. The sustained IC50 was derived from the current 400 msecs after voltage ramp from −80 mV to +30 mV. Pool #2 samples comprised di-PEGylated peptibodies and Pool #3 samples comprised mono-PEGylated peptibodies.

PEGylated Peptibody Pool # IC50 Sustained (nM) PEG-Fc-L10-SHK(2-35) 3 0.175(n = 4) PEG-Fc-L10-SHK(2-35) 2 0.158(n = 4) PEG-Fc-L10-OSK1 3 0.256(n = 3) PEG-Fc-L10-OSK1 2 0.332(n = 3)

Example 48 PEGylated Toxin Peptides

Shk and Osk-1 PEGylation, purification and analysis. Synthetic Shk or OSK1-1 toxin peptides were selectively PEGylated by reductive alkylation at their N-termini. Conjugation was achieved, with either Shk or OSK-1 toxin peptides, at 2 mg/ml in 50 mM NaH₂PO₄, pH 4.5 reaction buffer containing 20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDa monomethoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.). Conjugation reactions were stirred overnight at room temperature, and their progress was monitored by RP-HPLC. Completed reactions were quenched by 4-fold dilution with 20 mM NaOAc, pH 4, adjusted to pH 3.5 and chilled to 4° C. The PEG-peptides were then purified chromatographically at 4° C.; using SP Sepharose HP columns (GE Healthcare, Piscataway, N.J.) eluted with linear 0-1M NaCl gradients in 20 mM NaOAc, pH 4.0. (FIG. 68A and FIG. 68B) Eluted peak fractions were analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%. Principle contaminants observed were di-PEGylated toxin peptide and unmodified toxin peptide. Selected pools were concentrated to 2-5 mg/ml by centrifugal filtration against 3 kDa MWCO membranes and dialyzed into 10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then sterile filtered through 0.2 micron filters and purity determined to be >97% by SDS-PAGE and RP-HPLC (FIG. 69A and FIG. 69B). Reverse-phase HPLC was performed on an Agilent 1100 model HPLC running a Zorbax 5 μm 300SB-C8 4.6×50 mm column (Phenomenex) in 0.1% TFA/H₂0 at 1 ml/min and column temperature maintained at 40° C. Samples of PEG-peptide (20 μg) were injected and eluted in a linear 6-60% gradient while monitoring wavelengths 215 nm and 280 nm.

Electrophysiology performed by patch clamp on whole cells (see, Example 36) yielded a peak IC50 of 1.285 nM for PEG-OSK1 and 0.169 nM for PEG-ShK[1-35] (FIG. 74), in a concentration dependent block of the outward potassium current recorded from HEK293 cells stably expressing human Kv1.3 channel. The purified PEG-ShK[1-35] molecule, also referred to as “20K PEG-ShK[1-35]” and “PEG-ShK”, had a much longer half-life in vivo than the small ShK peptide (FIG. 59 and FIG. 60). PEG-ShK[1-35] suppressed severe autoimmune encephalomyelitis in rats (Example 45, FIGS. 61-63) and showed greater efficacy than the small native ShK peptide.

PEG conjugates of OSK1 peptide analogs were also generated and tested for activity in blocking T cell inflammation in the human whole blood assay (Example 46). As shown in Table 43, OSK1[Ala12], OSK1[Ala29], OSK1[1Nal11] and OSK1[Ala29, 1Nal34] analogs containing an N-terminal 20K PEG conjugate, all provided potent blockade of the whole blood cytokine response in this assay. The 20K PEG-ShK was also highly active (Table 43).

Example 49 Fc Loop Insertions of ShK and OSK1 Toxin Peptides

As exemplified in FIG. 70, FIG. 71, FIG. 72, and FIG. 73, disulphide-constrained toxin peptides were inserted into the human IgG1 Fc-loop domain, defined as the sequence D₁₃₇E₁₃₈L₁₃₉T₁₄₀K₁₄₁, according to the method published in Example 1 in Gegg et al., Modified Fc molecules, WO 2006/036834 A2 [PCT/US2005/034273]). Exemplary FcLoop-L2-OsK1-L2, FcLoop-L2-ShK-L2, FcLoop-L2-ShK-L4, and FcLoop-L4-OsK1-L2 were made having three linked domains. These were collected, purified and submitted for functional testing.

The peptide insertion for these examples was between Fc residues Leu₁₃₉ and Thr₁₄₀ and included 2-4 Gly residues as linkers flanking either side of the inserted peptide. However, alternate insertion sites for the human IgG1 Fc sequence, or different linkers, are also useful in the practice of the present invention, as is known in the art, e.g., as described in Example 13 of Gegg et al., Modified Fc molecules, WO 2006/036834 A2 [PCT/US2005/034273]).

Purified FcLoop OSK1 and FcLoop ShK1 molecules were tested in the whole blood assay of inflammation (see, Example 46). FcLoop-L2-OsK1-L2, FcLoop-L4-OsK1-L4 and FcLoop-L2-ShK-L2 toxin conjugates all provided potent blockade of the whole blood cytokine response in this assay, with IC50 values in the pM range (Table 43).

Example 50 Purification of ShK(2-35)-L-Fc from E. coli

Frozen, E. coli paste (117 g), obtained as described in Example 16 herein above, was combined with 1200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 ml water using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. 6.4 g of the pellet (total 14.2 g) was then dissolved in 128 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 120 ml of the pellet solution was then incubated with 0.67 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 5500 ml of the refolding buffer (3 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 2.5 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.

The refold was diluted with 5.5 L of water, and the pH was adjusted to 8.0 using acetic acid, then the solution was filtered through a 0.22 μm cellulose acetate filter and loaded on to a 35 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the Mab Select column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product immediately loaded on to a 5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (10 mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (10 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled and then applied to a 50 ml MEP Hypercel column (2.6 cm I.D.) at 10 ml/min in MEP Buffer A (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with a linear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH 4.0) followed by a step to 100% MEP Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the bulk of the desired product were pooled.

The MEP-pool was then concentrated to about 10 ml using a Pall Jumbo-Sep with a 10 kDa membrane. A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 76A). Then concentration of the material was determined to be 3.7 mg/ml using a calculated molecular mass of 30,253 and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 76B). The macromolecular state of the product was then determined using size exclusion chromatography on 70 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 76C). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 76D). The product was filtered through a 0.22 μm cellulose acetate filter and then stored at −80° C.

In Table 35, IC50 data for the purified E. coli-derived ShK[2-35]-L-Fc are compared to some other embodiments of the inventive composition of matter.

TABLE 35 E. coli-derived recombinant Fc-L-ShK[1-35], Fc-L-ShK[2-35], Fc-L-OSK1, Shk[1-35]-L-Fc and ShK[2-35]-L-Fc peptibodies containing Fc at either the N-terminus or C-termmus show potent blockade of human Kv1.3. Kv1.3 IC₅₀ Kv1.3 IC₅₀ Molecule by WCVC (nM) by IWQ (nM) E. coli-derived Fc-L-ShK[1-35] 1.4 E. coli-derived Fc-L-ShK[2-35] 1.3 2.8 E. coli-derived Fc-L-OSK 1 3.2 E. coli-derived Shk[1-35]-L-Fc 2.4 E. coli-derived ShK[2-35]-L-Fc 4.9 CHO-derived Fc-L10-ShK[1-35] 2.2 R1Q The activity of the CHO-derived Fc-L10-ShK[1-35] R1Q mutant is also shown. Whole cell patch clamp electrophysiology (WCVC), by methods described in Example 36, was performed using HEK293/Kv1.3 cells and the IC50 shown is the average from dose-response curves from 3 or more cells. IonWorks ™ (IWQ) planar patch clamp electrophysiology by methods described in Example 44 was on CHO/Kv1.3 cells and the average IC50 is shown. The inventive molecules were obtained by methods as described in the indicated Example: E. coli-derived Fc-L-ShK[1-35] (Example 3 and Example 38), E. coli-derived Fc-L-ShK[2- 35] (Example 4 and Example 39), E. coli Fc-L-OSK1 (Example 10 and Example 40), ShK[1-35]-L-Fc (Example 15 and Example 51), and ShK[2-35]-L-Fc (Example 16 and this Example 50). CHO-derived Fc-L10-ShK[1-35] R1Q molecule was generated using methods similar to those described for CHO-derived Fc-L10-ShK[1-35].

Example 51 Purification of Met-ShK(1-35)-Fc from E. coli

Frozen, E. coli paste (65 g), obtained as described in Example 15 herein above was combined with 660 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 660 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 660 ml water using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. 13 g of the pellet was then dissolved in 130 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 10 ml of the pellet solution was then incubated with 0.1 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 1000 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.

The refold was diluted with 1 L of water, and filtered through a 0.22 μm cellulose acetate filter then loaded on to a 35 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the Mab Select column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product immediately loaded on to a 5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (20 mM NaH₂PO₄, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (20 mM NaH₂PO₄, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled.

The S-pool was then concentrated to about 10 ml using a Pall Jumbo-Sep with a 10 kDa membrane. A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 77A). Then concentration of the material was determined to be 3.1 mg/ml using a calculated molecular mass of 30,409 and extinction coefficient of 36,900 M⁻¹ cm⁻¹. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 77B). The macromolecular state of the product was then determined using size exclusion chromatography on 93 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 77C). The product was then subject to mass spectral analysis by MALDI mass spectrometry.

An aliquot of the sample was spotted with the MALDI matrix sinapinic acid on sample plate. A Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse) was used to collect spectra. The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots (FIG. 77D). External mass calibration was accomplished using purified proteins of known molecular masses.

The IC₅₀ for blockade of human Kv1.3 by purified E. coli-derived Met-ShK(1-35)-Fc, also referred to as “ShK[1-35]-L-Fc”, is shown in Table 35 herein above.

Example 52 Bacterial Expression of OsK1-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21amgR-pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of OsK1-L-Fc. Oligos used to form duplex are shown below:

GGGTGTTATCATCAACGTTAAATGCAAAAT SEQ ID NO: 1347 CTCCCGTCAGTGCCTGGAACCGTGCA AAAAAGCTGGTATGCGT//; TTCGGTAAATGCATGAACGGTAAATGCCA SEQ ID NO: 1348 CTGCACCCCGAAATCTGGTGG TGGTGGTTCT//; CACCAGAACCACCACCACCACCAGATTTCG SEQ ID NO: 1349 GGGTGCAGTGGCATTTACCGTTCA TGCATTTACCGAAACGCAT//; ACCAGCTTTTTTGCACGGTTCCAGGCACT SEQ ID NO: 1310 GACGGGAGATTTTGCATTTAA CGTTGATGATAAC//;

The oligos shown above were used to form the duplex shown below:

GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCGTGCAAAAA   1 ---------+---------+---------+---------+---------+---------+  60     CAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGTCACGGACCTTGGCACGTTTTT  G  V  I  I  N  V  K  C  K  I  S  R  Q  C  L  E  P  C  K  K — AGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAATCTGG  61 ---------+---------+---------+---------+---------+---------+ 120 TCGACCATACGCAAAGCCATTTACGTACTTGCCATTTACGGTGACGTGGGGCTTTAGACC  A  G  M  R  F  G  K  C  M  N  G  K  C  H  C  T  P  K  S  G — TGGTGGTGGTTCT //SEQ ID NO: 1350 121 ---------+------- 137 ACCACCACCAAGACCAC //SEQ ID NO: 1352  G  G  G  S  G — //SEQ ID NO: 1351

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 53 Bacterial Expression of Gly-ShK(1-35)-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21amgR-pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Gly-ShK(1-35)-L-Fc. Oligos used to form duplex are shown below:

GGGTCGTTCTTGTATTGATACTATTCCA SEQ ID NO: 1313 AAATCTCGTTGTACTGCTTTTCA ATGTAAACATTCTATGAAATAT CGTCTTTCTT//; TTTGTCGTAAAACTTGTGGTACTTGTTCTG SEQ ID NO: 1314 GTGGTGGTGGTTCT//; CACCAGAACCACCACCACCAGAACAAG SEQ ID NO: 1353 TACCACAAGTTTTACGACAAA AAGAAAGACGATATTTCATAGA ATGTTTACATTGA//; AAAGCAGTACAACGAGATTTTGGAATA SEQ ID NO: 1354 GTATCAATACAAGAACG//

The oligos shown above were used to form the duplex shown below:

GGGTCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACA   1 ---------+---------+---------+---------+---------+---------+  60     GCAAGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGT  G  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H — TTCTATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGCTACTTGTTCTGGTGGTGG  61 ---------+---------+---------+---------+---------+---------+ 120 AAGATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACC  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G — TGGTTCT //SEQ ID NO: 1355 121 ---------+- 131 ACCAAGACCAC //SEQ ID NO: 1357  G  S  G — //SEQ ID NO: 1356

Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.

Example 54 Bacterial Expression of CH2-OSK1 Inhibitor of Kv1.3

The methods to clone and express the fusion of a CH2 domain of an Fc with OSK1 in bacteria were generally as described in Example 3. The vector used was pAMG21.G2.H6.G3.CH2.(G4S)2.OSK. Briefly, the pAMG21 vector was modified to remove the multi-cloning site's BamHI. This allowed the BamHI in front of the OSK as a site to swap out different sequences for fusion with the OSK. The sequence upstream of the OSK1 coding sequence was ligated between the NdeI and BamHI sites.

The sequence of the entire vector, including the insert was the following:

SEQ ID NO: 4914 gtcgtcaacgaccccccattcaagaacagcaagcagcattgagaactttg gaatccagtccctcttccacctgctgaccggatcagcagtccccggaaca tcgtagctgacgccttcgcgttgctcagttgtccaaccccggaaacggga aaaagcaagttttccccgctcccggcgtttcaataactgaaaaccatact atttcacagtttaaatcacattaaacgacagtaatccccgttgatttgtg cgccaacacagatcttcgtcacaattctcaagtcgctgatttcaaaaaac tgtagtatcctctgcgaaacgatccctgtttgagtattgaggaggcgaga tgtcgcagacagaaaatgcagtgacttcctcattgagtcaaaagcggttt gtgcgcagaggtaagcctatgactgactctgagaaacaaatggccgttgt tgcaagaaaacgtcttacacacaaagagataaaagtttttgtcaaaaatc ctctgaaggatctcatggttgagtactgcgagagagaggggataacacag gctcagttcgttgagaaaatcatcaaagatgaactgcaaagactggatat actaaagtaaagactttactttgtggcgtagcatgctagattactgatcg tttaaggaattttgtggctggccacgccgtaaggtggcaaggaactggtt ctgatgtggatttacaggagccagaaaagcaaaaaccccgataatcttct tcaacttttgcgagtacgaaaagattaccggggcccacttaaaccgtata gccaacaattcagctatgcggggagtatagttatatgcccggaaaagttc aagacttctttctgtgctcgctccttctgcgcattgtaagtgcaggatgg tgtgactgatcttcaccaaacgtattaccgccaggtaaagaacccgaatc cggtgtttacaccccgtgaaggtgcaggaacgctgaagttctgcgaaaaa ctgatggaaaaggcggtgggcttcacttcccgttttgatttcgccattca tgtggcgcacgcccgttcgcgtgatctgcgtcgccgtatgccaccagtgc tgcgtcgtcgggctattgatgcgctcttgcaggggctgtgtttccactat gacccgctggccaaccgcgtccagtgctccatcaccacgctggccattga gtgcggactggcgacggagtctgctgccggaaaactctccatcacccgtg ccacccgtgccctgacgttcctgtcagagctgggactgattacctaccag acggaatatgacccgcttatcgggtgctacattccgaccgatatcacgtt cacatctgcactgtttgctgccctcgatgtatcagaggaggcagtggccg ccgcgcgccgcagccgtgtggtatgggaaaacaaacaacgcaaaaagcag gggctggataccctgggcatggatgaactgatagcgaaagcctggcgttt tgttcgtgagcgttttcgcagttatcagacagagcttaagtcccgtggaa taaagcgtgcccgtgcgcgtcgtgatgcggacagggaacgtcaggatatt gtcaccctggtgaaacggcagctgacgcgcgaaatcgcggaagggcgctt cactgccaatcgtgaggcggtaaaacgcgaagttgagcgtcgtgtgaagg agcgcatgattctgtcacgtaaccgtaattacagccggctggccacagct tccccctgaaagtgacctcctctgaataatccggcctgcgccggaggctt ccgcacgtctgaagcccgacagcgcacaaaaaatcagcaccacatacaaa aaacaacctcatcatccagcttctggtgcatccggccccccctgttttcg atacaaaacacgcctcacagacggggaattttgcttatccacattaaact gcaagggacttccccataaggttacaaccgttcatgtcataaagcgccat ccgccagcgttacagggtgcaatgtatcttttaaacacctgtttatatct cctttaaactacttaattacattcatttaaaaagaaaacctattcactgc ctgtccttggacagacagatatgcacctcccaccgcaagcggcgggcccc taccggagccgctttagttacaacactcagacacaaccaccagaaaaacc ccggtccagcgcagaactgaaaccacaaagcccctccctcataactgaaa agcggccccgccccggtccgaagggccggaacagagtcgcttttaattat gaatgttgtaactacttcatcatcgctgtcagtcttctcgctggaagttc tcagtacacgctcgtaagcggccctgacggcccgctaacgcggagatacg ccccgacttcgggtaaaccctcgtcgggaccactccgaccgcgcacagaa gctctctcatggctgaaagcgggtatggtctggcagggctggggatgggt aaggtgaaatctatcaatcagtaccggcttacgccgggcttcggcggttt tactcctgtttcatatatgaaacaacaggtcaccgccttccatgccgctg atgcggcatatcctggtaacgatatctgaattgttatacatgtgtatata cgtggtaatgacaaaaataggacaagttaaaaatttacaggcgatgcaat gattcaaacacgtaatcaatatcgggggtgggcgaagaactccagcatga gatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccg aagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatgg caggttgggcgtcgcttggtcggtcatttcgaaccccagagtcccgctca gaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggag cggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagc tcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgc cacacccagccggccacagtcgatgaatccagaaaagcggccattttcca ccatgatattcggcaagcaggcatcgccatgagtcacgacgagatcctcg ccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgag cccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttcca tccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatggg caggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgat ggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccg gcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcg agcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgc tgcctcgtcctgcaattcattcaggacaccggacaggtcggtcttgacaa aaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagag cagccgattgtctgttgtgcccagtcatagccgaatagcctctccaccca agcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacg atcctcatcctgtctcttgatctgatcttgatcccctgcgccatcagatc cttggcggcaagaaagccatccagtttactttgcagggcttcccaacctt accagagggcgccccagctggcaattccggttcgcttgctgtccataaaa ccgcccagtctagctatcgccatgtaagcccactgcaagctacctgcttt ctctttgcgcttgcgttttcccttgtccagatagcccagtagctgacatt catccggggtcagcaccgtttctgcggactggctttctacgtgttccgct tcctttagcagcccttgcgccctgagtgcttgcggcagcgtgaagctaca tatatgtgatccgggcaaatcgctgaatattccttttgtctccgaccatc aggcacctgagtcgctgtctttttcgtgacattcagttcgctgcgctcac ggctctggcagtgaatgggggtaaatggcactacaggcgccttttatgga ttcatgcaaggaaactacccataatacaagaaaagcccgtcacgggcttc tcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgc tgttcagcagttcctgccctctgattttccagtctgaccacttcggatta tcccgtgacaggtcattcagactggctaatgcacccagtaaggcagcggt atcatcaacaggcttacccgtcttactgtcgaagacgtgcgtaacgtatg catggtctccccatgcgagagtagggaactgccaggcatcaaataaaacg aaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcgg tgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgtt gcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgc caggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgt ttctacaaactcttttgtttatttttctaaatacattcaaatatggacgt cgtacttaacttttaaagtatgggcaatcaattgctcctgttaaaattgc tttagaaatactttggcagcggtttgttgtattgagtttcatttgcgcat tggttaaatggaaagtgaccgtgcgcttactacagcctaatatttttgaa atatcccaagagctttttccttcgcatgcccacgctaaacattctttttc tcttttggttaaatcgttgtttgatttattatttgctatatttatttttc gataattatcaactagagaaggaacaattaatggtatgttcatacacgca tgtaaaaataaactatctatatagttgtctttctctgaatgtgcaaaact aagcattccgaagccattattagcagtatgaatagggaaactaaacccag tgataagacctgatgatttcgcttctttaattacatttggagatttttta tttacagcattgttttcaaatatattccaattaatcggtgaatgattgga gttagaataatctactataggatcatattttattaaattagcgtcatcat aatattgcctccattttttagggtaattatccagaattgaaatatcagat ttaaccatagaatgaggataaatgatcgcgagtaaataatattcacaatg taccattttagtcatatcagataagcattgattaatatcattattgcttc tacaggctttaattttattaattattctgtaagtgtcgtcggcatttatg tctttcatacccatctctttatccttacctattgtttgtcgcaagttttg cgtgttatatatcattaaaacggtaatagattgacatttgattctaataa attggatttttgtcacactattatatcgcttgaaatacaattgtttaaca taagtacctgtaggatcgtacaggtttacgcaagaaaatggtttgttata gtcgattaatcgatttgattctagatttgttttaactaattaaaggagga ataacatatgggcggccatcatcatcatcatcatggcgggggaccgtcag ttttcctcttccccccaaaacccaaggacaccctcatgatctcccggacc cctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggt caagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctc accgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggt ctccaacaaagccctcccagcccccatcgagaaaaccatctccggcggcg gcggcagcggcggcggcggatccggtgttatcatcaacgttaaatgcaaa atctcccgtcagtgcctggaaccgtgcaaaaaagctggtatgcgtttcgg taaatgcatgaacggtaaatgccactgcaccccgaaataatgaattcgag ctcactagtgtcgacctgcagggtaccatggaagcttactcgaagatccg cggaaagaagaagaagaagaagaaagcccgaaaggaagctgagttggctg ctgccaccgctgagcaataactagcataaccccttggggcctctaaacgg gtcttgaggggttttttgctgaaaggaggaaccgctcttcacgctcttca cgcggataaataagtaacgatccggtccagtaatgacctcagaactccat ctggatttgttcagaacgctcggttgccgccgggcgttttttattggtga gaatcgcagcaacttgtcgcgccaatcgagccatgtc//.

The insert DNA sequence was the following:

atgggcggccatcatcatcatcatcatggcgggg SEQ ID NO: 4915 gaccgtcagttttcctcttccccccaaaacccaa ggacaccctcatgatctcccgga cccctgaggtcacatgcgtggtggtggacgtgag ccacgaagaccctgaggtcaagttcaactggtac gtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagt acaacagcacgtaccgtgtggtcagcgtcctcac cgtcctgcaccaggactggctg aatggcaaggagtacaagtgcaaggtctccaac aaagccctcccagcccccatcgagaaaaccatc tccggcggcggcggcagcgg cggcggcggatccggtgttatcatcaacgttaa atgcaaaatctcccgtcagtgcctggaaccgtgc aaaaaagctggtatgcgtttcggt aaatgcatgaacggtaaatgccactgcaccccg aaa//.

The amino acid sequence of the CH2-OSK1 fusion protein product was the following:

MGGHHHHHHGGGPSVFLFPPKPKDTLMIS SEQ ID NO: 4917 RTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISGGGGSG GGGSGVIINVKCKISRQCLEPCKKAGMRFGK CMNGKCHCTPK//.

SEQ ID NO: 4917 includes the OSK1 sequence

GVIINVKCKISRQCLEPCKKAGMRFGKCMNG (SEQ ID NO: 25) KCHCTPK.

Purification and refolding of CH2-OSK1 expressed in bacteria. Frozen, E. coli paste (13.8 g) was combined with 180 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 50 min at 4° C. The pellet was then resuspended in 90 ml 1% deoxycholate using a tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C. The pellet was then resuspended in 90 ml water using a tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C. The pellet (3.2 g) was then dissolved in 64 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The suspension was then incubated at room temperature (about 23° C.) for 30 min with gentle agitation followed by centrifugation at 15,300 g for 30 min at 4° C. The supernatant (22 ml) was then reduced by adding 220 μl 1 M dithiothreitol and incubating at 37° C. for 30 minutes. The reduced suspension (20 ml) was transferred to 2000 ml of the refolding buffer (1 M urea, 50 mM ethanolamine, 160 mM arginine HCl, 0.02% NaN₃, 1.2 mM cystamine HCl, 4 mM cysteine, pH 9.8) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for approximately 2.5 days at 4° C.

Ten milliliters of 500 mM imidazole was added to the refolding solution and the pH was adjusted to pH to 8.0 with 5 M acetic acid. The refold was then filtered through a 0.45 μm cellulose acetate filter with two pre-filters. This material was then loaded on to a 50 ml Qiagen Ni-NTA Superflow column (2.6 cm ID) in Ni-Buffer A (50 mM NaH₂PO₄, 300 mM NaCl, pH 7.5) at 15 ml/min 13° C. The column was then washed with 10 column volumes of Ni-Buffer A followed by 8% Ni-Buffer B (250 mM imidazole, 50 mM NaH₂PO₄, 300 mM NaCl, pH 7.5) at 25 ml/min. The column was then eluted with 60% Ni-Buffer B followed by 100% Ni-Buffer B at 10 ml/min. The peak fractions were collected and dialyzed against S-Buffer A (10 mM NaH₂PO₄, pH 7.1)

The dialyzed sample was then loaded on to a 5 ml Amersham SP-HP HiTrap column at 5 ml/min in S-Buffer A at 13° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (10 mM NaH₂PO₄, 1 M NaCl, pH 7.1) followed by a step to 100% S-Buffer B at 1.5 ml/min 13° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pool was then concentrated to about 1.6 ml using a Pall Macrosep with a 10 kDa membrane at 4° C. The concentrated sample was then filtered through a 0.22 μm cellulose acetate centrifugal filter.

A spectral scan was then conducted on 10 μl of the combined pool diluted in 150 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 78). The concentration of the filtered material was determined to be 3.35 mg/ml using a calculated molecular mass of 17,373 g/mol and extinction coefficient of 17,460 M⁻¹ cm⁻¹. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 79). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH₂PO₄, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 80). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C₄, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 81). The product was then stored at −80° C.

PEGylation of CH2-OSK1. The CH2-OSK1 fusion protein was diluted to 2 mg/ml in 50 mM sodium acetate, 10 mM sodium cyanoborohydride, pH 4.8 with a 4-fold molar excess of 20 kD methoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.). The reaction was allowed to proceed overnight (˜18 hrs) at 4° C. Upon completion, reaction was quenched with 4 volumes of 10 mM sodium acetate, 50 mM NaCl, pH 5, then loaded at 0.7 mg protein/ml resin to an SP Sepharose HP column (GE Healthcare, Piscataway, N.J.) equilibrated in 10 mM sodium acetate, 50 mM NaCl, pH 5. The mono-PEGylated CH2-Osk fusion was eluted with a linear 50 mM-1 M NaCl gradient (FIG. 82). Peak fractions were evaluated by SDS-PAGE and the mono-PEG-CH2-OSK1 fractions pooled, concentrated and dialyzed into Dulbecco's Phosphate Buffered Saline. The final product was analyzed by SDS-PAGE (FIG. 83).

As shown in Table 43, the purified CH2-OSK1 (“L2-6H-L3-CH2-L10-OsK1(1-38)”) and PEGylated CH2-OSK1 (“20 k PEG-L2-6H-L3-CH2-L10-OSK1(1-38)”) molecules were active in blocking inflammation in the human whole blood assay (See, Example 46).

Example 55 Bioactivity of OSK1 Peptide Analogs

The activity of OSK1 peptide analogs in blocking human Kv1.3 versus human Kv1.1 current is shown in FIGS. 84 through 86 and Tables 37-41. Three electrophysiology techniques were used (See, e.g., Example 36 and Example 44). Whole cell patch clamp (FIGS. 84 & 85 and Table 41) represents a low throughput technique which is well established in the field and has been available for many years. We also used two new planar patch clamp techniques, PatchXpress and IonWorks Quattro, with improved throughput which facilitate assessment of potency and selectivity of novel OSK1 analogs described in this application. The PatchXpress technique is of moderate throughput and the novel OSK1[Ala-12] analog (SEQ ID No:1410) had similar Kv1.3 potency and selectivity over Kv1.1 to that observed by whole cell patch clamp (FIG. 84 and Table 41). IonWorks Quattro represents a 384-well planar patch clamp electrophysiology system of high throughput. Using this IonWorks system, the novel OSK1[Ala-29] analog (SEQ ID No:1424) showed potent inhibition of the Kv1.3 current and improved selectivity over Kv1.1 (FIG. 86). The OSK1[Ala-29] analog (SEQ ID No:1424) showed similar Kv1.3 activity and selectivity over Kv1.1 by whole cell patch clamp electrophysiology (FIG. 85) to that observed by IonWorks (FIG. 86). The Kv1.3 and Kv1.1 activities of Alanine, Arginine, Glutamic acid and 1-Naphthylalanine analogs of OSK1 were determined by IonWorks electrophysiology and is reported in Tables 37-40. OSK1 peptide analogs identified by IonWorks to have good potency or Kv1.3 selectivity, were tested further in whole cell patch clamp studies (see, Table 41). The Kv1.3 IC50 of the His34Ala analog of OSK1 (SEQ ID No:1428) was 797 fold lower than its IC50 against Kv1.1 (Table 41), demonstrating that this analog is a highly selective Kv1.3 inhibitor. In this same assay, native OSK1 (SEQ ID NO: 25) showed only slight Kv1.3 selectivity, with the Kv1.1 IC50 being only 5 fold higher than Kv1.3.

The novel OSK1 peptide analogs described in this application which inhibit Kv1.3 are useful in the treatment of autoimmune disease and inflammation. Kv1.3 is expressed on T cells and Kv1.3 inhibitors suppress inflammation by these cells. As one measure of inflammation mediated by T cells, we examined the impact of OSK1 analogs on IL-2 and IFN-g production in human whole blood following addition of a pro-inflammatory stimulus (Tables 36-40 and Table 42). “WB/IL-2” in these tables refers to the assay measuring IL-2 response of whole blood (see, Example 46), whereas “WB/IFNg” refers to the assay measuring IFNg response of whole blood (see, Example 46). The IC50 values listed in Tables 36-40 and 42, represent the average IC50 value determined from experiments done with two or more blood donors. The whole blood assay (see Example 46) allows for a combined measurement of the potency of the analogs in blocking inflammation and Kv1.3, as well as an assessment of the stability of the molecules in a complex biological fluid. Using this assay, several OSK1 analogs were examined and found to potently suppress inflammation (FIGS. 90C & 90D, Tables 36-40). Some of these analogs showed reduced activity in this whole blood assay, which may indicate that these residues play an important role in binding Kv1.3. Relative to the immunosuppressive agent cyclosporin A, Kv1.3 peptide inhibitors ShK-Ala22, OSK1-Ala29, and OSK1-Ala12 were several orders of magnitude more potent in blocking the cytokine response in human whole blood (FIG. 90).

The solution NMR structure of OSK1 has been solved and is provided as pdb accession number “1SCO” in Entrez's Molecular Modeling Database (MMDB) [J. Chen et al. (2003) Nucleic Acids Res. 31, 474-7]. FIG. 89 shows space filling (FIG. 89A, 89B, 89D) and worm (FIG. 89C) Cn3D rendering of the OSK1 structure. Light colored OSK1 amino acid residues Phe25, Gly26, Lys27, Met29 and Asn30 are shown in FIG. 89B. Some analogs of these residues were found to significantly reduce Kv1.3 activity (Tables 37-40), implying that these residues may make important contacts with the Kv1.3 channel. The molecular structure shown in FIG. 89A indicates these amino acids reside on a common surface of the OSK1 three-dimensional (3D) structure. FIG. 89D shows OSK1 residues (light shading) Ser11, Met29 and His34. These residues when converted to some amino acid analogs, provide improved Kv1.3 selectivity over Kv1.1 (Table 41). Although about 23 amino acids are between residues Ser11 and His34 in the contiguous polypeptide chain, the structure shown in FIG. 89D illustrates that in the 3D structure of the folded molecule these residues are relatively close to one another. Upon comparing FIGS. 89D and 89B, one can see that residues His34 and Ser11 (FIG. 89D) are on the left and right side, respectively, and adjacent to the major Kv1.3 contact surface displayed in FIG. 89B. It is envisioned that molecular modeling can be used to identify OSK1 analogs with improved Kv1.3 activity and selectivity, upon considering the Kv1.3 and Kv1.1 bioactivity information provided in Tables 37 through 42 and the solution NMR structure of OSK1 described above. FIG. 89C shows a worm rendering of the OSK1 structure with secondary structure elements (beta strands and alpha helices) depicted. The primary amino acid sequence of OSK1 is provided in FIG. 89E and amino acid residues comprising the beta strands & alpha helix are underlined. Wiggly lines in FIG. 89C indicate amino acid residues between or beyond these secondary structure elements, whereas straight lines depict the three disulfide bridges in OSK1. The first beta strand (β1) shown in FIGS. 89C and 89E contains no disulfide bridges to link it covalently to other secondary structure elements of the OSK1 molecule, unlike beta strand 3 (β3) that has two disulfide bridges with the alpha helix (α1). As shown in Table 42, OSK1 analogs without beta strand 1 (labeled “des 1-7”) still retain activity in blocking inflammation (see SEQ ID No: 4989 of Table 42) suggesting that this region of OSK1 is not essential for the molecules Kv1.3 bioactivity.

OSK1 analogs containing multiple amino acid changes were generated and their activity in the human whole blood assay of inflammation is provided in Table 42. Several analogs retain high potency in this assay despite as many as 12 amino acid changes. Based on the improved Kv1.3 selectivity of analogs with single amino acid changes, anologs with multiple amino acid changes may result in additional improvements in selectivity. It is also envisioned that analogs with multiple amino acid changes may have improved activity or stability in vivo, alone or in the context of a peptide conjugate to a half-life prolonging moiety.

Kv1.3 peptide toxins conjugated to half-life prolonging moieties are provided within this application. The bioactivity of several toxin conjugates is described in Table 43. OSK1 analogs with a N-terminal half-life prolonging 20K PEG moiety (see Example 48) were found to provide potent suppression of the whole blood IL-2 (“WB/IL-2”) and IFNg (“WB/IFNg”) response (Table 43). The 20K PEG-ShK conjugate, shown earlier to have prolonged half-life in vivo (see Examples 44 and 48), was also highly active in this whole blood assay. The FcLoop-OSK1 conjugates (see Example 49) were highly active in blocking inflammation (Table 43), and the CH2-OSK1 or PEG-CH2-OSK1 conjugates (see Example 54) provided modest blockade of the whole blood cytokine response (Table 43). The IL-2 and IFNg cytokine response measured in this whole blood assay results from T cell activation. Since this cytokine response is Kv1.3 dependent and potently blocked by the Kv1.3 peptide and peptide-conjugate inhibitors described herein, these whole blood studies illustrate the therapeutic utility of these molecules in treatment of immune disorders.

TABLE 36 Activity of OSK1 analogs in blocking thapsigargin-induced IL-2 and IFNg production in 50% human whole blood as described in Example 46. Thapsigargin Induced IL-2 & IFNg in Human Whole Blood Average Std Dev Average Std Dev Analog IC₅₀ Divided IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) by Ala-1 IC₅₀ OSK1 Analog IL-2 IL-2 IFNg IFNg IL2 IFNg Ala-1 (SEQ ID No: 1400) 0.1220 0.0791 0.1194 0.0802 1.00 1.00 Ala-2 (SEQ ID No: 1401) 0.0884 0.0733 0.1035 0.0776 0.72 0.87 Ala-3 (SEQ ID No: 1402) 0.0883 0.0558 0.0992 0.1007 0.72 0.83 Ala-4 (SEQ ID No: 1403) 0.1109 0.1098 0.0873 0.0993 0.91 0.73 Ala-5 (SEQ ID No: 1404) 0.0679 0.0566 0.0670 0.0446 0.56 0.56 Ala-6 (SEQ ID No: 1405) 0.0733 0.0477 0.0805 0.0696 0.60 0.67 Ala-7 (SEQ ID No: 1406) 0.0675 0.0383 0.0591 0.0260 0.55 0.49 Ala-9 (SEQ ID No: 1407) 0.0796 0.0761 0.0711 0.0627 0.65 0.60 Ala-10 (SEQ ID No: 1408) 0.0500 0.0425 0.0296 0.0084 0.41 0.25 Ala-12 (SEQ ID No: 1410) 0.1235 0.0823 0.1551 0.0666 1.01 1.30 Ala-13 (SEQ ID No: 1411) 0.1481 0.0040 0.1328 0.0153 1.21 1.11 Ala-15 (SEQ ID No: 1412) 0.1075 0.1075 0.88 0.90 Ala-16 (SEQ ID No: 1413) 0.1009 0.1009 0.83 0.84 Ala-17 (SEQ ID No: 1414) 0.1730 0.1730 1.42 1.45 Ala-19 (SEQ ID No: 1415) 0.1625 0.1625 1.33 1.36 Ala-20 (SEQ ID No: 1416) 0.3790 0.3790 3.11 3.17 Ala-22 (SEQ ID No: 1418) 7.0860 7.0860 58.07 59.33 Ala-23 (SEQ ID No: 1419) 0.2747 0.2747 2.25 2.30 Ala-25 (SEQ ID No: 1421) 3.0800 3.0800 25.24 25.79 Ala-27 (SEQ ID No: 1423) 3.4510 2.5781 1.5792 1.9217 28.28 13.22 Ala-29 (SEQ ID No: 1424) 0.4469 0.1727 0.2919 0.2422 3.66 2.44 Ala-3O (SEQ ID No: 1425) 0.9710 0.7538 0.6370 0.2674 7.96 5.33 Ala-34 (SEQ ID No: 1428) 0.0725 0.0275 0.0573 0.0341 0.59 0.48 Pro-12, Lys-16, Asp-20, Ile- 0.5138 0.4064 0.5127 0.1597 4.21 4.29 23, Ile-29, Ala-34 (SEQ ID No: 1393)

TABLE 37 OSK1 Alanine Analogs. SEQ ID Analogue Activity (IC50, pM) NO: Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1400 G1A 41.11 13.89 122.035 119.425 1401 V2A 81.78 9.94 88.395 103.515 1402 I3A 96.59 10.64 88.255 99.16 1403 I4A 195.30 16.92 110.865 87.255 1404 N5A 159.98 14.01 67.91 66.985 1405 V6A 173.75 12.84 73.26 80.465 1406 K7A 181.04 21.88 67.5 59.075 1407 K9A 166.27 40.59 79.58 71.065 1408 I10A 91.23 4.46 49.97 29.63 1409 S11A 40.79 113.15 90 110 1410 R12A 389.90 55.89 123.49 155.1 1411 Q13A 249.45 21.65 148.05 132.75 1412 L15A 43.07 15.04 107.5 107.5 1413 E16A 21.55 6.87 100.9 100.9 1414 P17A 33.89 9.08 173 173 1415 K19A 210.48 16.85 162.5 162.5 1416 K20A 1036.08 185.01 379 379 1417 1418 G22A >3000 >3000 7086 7086 1419 M23A 71.39 38.63 274.7 274.7 1420 R24A >3000 1890.78 1421 F25A 1486.97 47.30 3080 3080 1422 G26A 710.98 733.36 12075 10730 1423 K27A 232.44 >3000 1232 1579.15 1424 M29A 59.47 >3333 446.9 291.85 1425 N30A 692.54 >3000 971 637 1426 G31A 70.17 61.78 1427 K32A 41.3 34 1428 H34A 19.36 368.41 72.54 57.29 1429 T36A 728.4 723.5 1430 P37A 956 849.7 1431 K38A 221 343

TABLE 38 OSK1 Arginine Analogs. SEQ ID Analogue Activity (IC50, pM) NO: Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1432 G1R 68.75 9.91 554 991 1433 V2R 133.34 25.79 775 986 1434 I3R 19.90 2.47 148 180 1435 I4R 10.41 1.92 168 175 1436 N5R 13.62 2.15 95 120 1437 V6R 8.65 2.40 84 115 1438 K7R 13.17 <1.52401 78 71 1439 K9R 11.99 2.01 107 77 1440 I10R 11.68 1.73 307 474 1441 S11R 16.72 210.05 2118 4070 1442 Q13R 15.34 <1.52401 160 172 1443 L15R 13.73 2.16 93 116 1444 E16R 10.36 <1.52401 556 454 1445 P17R 10.42 <1.52401 202 355 1446 K19R 12.57 2.41 44 62 1447 K20R 9.85 <1.52401 67 83 1448 A21R 14.92 2.61 90 149 1449 G22R 23.74 3.49 292 349 1450 M23R 12.34 2.01 182 148 1451 F25R >3333 817.42 25027 30963 1452 G26R >3333 >3333 100000 100000 1453 K27R 1492.94 >3333 15088 10659 1454 M29R 200.39 1872.11 11680 7677 1455 N30R 18.90 45.71 405 445 1456 G31R 22.16 1.59 314 343 1457 K32R 30.83 7.24 28 34 1458 H34R 13.57 4.49 92 108 1459 T36R 1308.07 26.55 9697 10050 1460 P37R 13.32 2.01 229 253 1461 K38R 14.99 1.84 39 40

TABLE 39 OSK1 Glutamic Acid Analogs. SEQ ID Analogue Activity (IC50, pM) NO: Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1462 G1E 185.78 50.97 1217 1252 1463 V2E 36.23 35.01 97 184 1464 I3E 22.00 42.99 120 160 1465 I4E 15.65 3.19 218 191 1466 N5E 23.38 4.44 100 65 1467 V6E 17.73 2.43 48 68 1468 K7E 14.16 <1.52401 58 68 1469 K9E 31.76 110.67 179 171 1470 I10E 120.35 33.50 2573 2736 1471 S11E >3333 >3333 39878 16927 1472 R12E 89.71 193.25 1787 2001 1473 Q13E 45.87 6.28 1063 799 1474 L15E 47.48 436.05 785 1059 1475 P17E 14.47 1.81 520 947 1476 K19E 23.51 13.71 1477 K20E 25.45 5.76 1478 A21E 7.37 <1.52401 117 138 1479 G22E 13.88 2.56 109 164 1480 M23E 24.28 10.44 606 666 1481 R24E 7161 9543 1482 F25E >3333 >3333 100000 100000 1483 G26E >3333 >3333 100000 100000 1484 K27E >3333 548.55 5548 7144 1485 M29E >3333 >3333 27099 24646 1486 N30E 14024 24372 1487 G31E 12.01 2.37 95 111 1488 K32E 15.56 17.31 62 63 1489 H34E 330.15 1689.82 1618 2378 1490 T36E 161.06 >3333 1742 2420 1491 P37E 62.67 622.18 239 1604 1492 K38E 25.76 34.33 526 713

TABLE 40 OSK1 Naphthylalanine Analogs. SEQ ID Analogue Activity (IC50, pM) NO: Analogue Kv1.3 Kv1.1 WB/IL-2 WB/IFNg 1493 G1Nal 20.66 33.93 2793 2565 1494 V2Nal 11.55 2.46 750 524 1495 I3Nal 10.31 2.34 907 739 1496 I4Nal 15.03 <1.52401 1094 1014 1497 N5Nal 21.78 <1.52401 760 431 1498 V6Nal 20.97 <1.52401 1776 2465 1499 K7Nal 23.61 <1.52401 222 246 1500 K9Nal 65.82 2.92 1070 1217 1501 I10Nal 45.44 <1.52401 184 257 1502 S11Nal 95.87 >3333 23915 17939 1503 R12Nal 37.66 24.99 460 387 1504 Q13Nal 13.44 <1.52401 140 198 1505 L15Nal 17.84 <1.52401 358 370 1506 E16Nal 9.58 <1.52401 1025 1511 1507 P17Nal 16.19 <1.52401 193 357 1508 K19Nal 17.22 <1.52401 58 99 1509 K20Nal 13.53 <1.52401 74 12 1510 A21Nal 26.10 <1.52401 315 434 1511 G22Nal >3333 426.27 10328 10627 1512 H23Nal 35.96 64.37 581 1113 1513 R24Nal 45.26 2.85 293 818 1514 F25Nal 28.63 51.75 1733 1686 1515 G26Nal >3333 1573.75 9898 10651 1516 K27Nal >3333 1042.84 14971 27025 1517 M29Nal 93.46 46.88 100000 100000 1518 N30Nal >3333 1283.88 100000 37043 1519 G31Nal 33.76 <1.52401 331 467 1520 K32Nal 26.13 1.91 134 196 1521 H34Nal 60.31 >3333 3323 6186 1522 T36Nal 100000 37811 1523 P37Nal 70.80 6.74 1762 3037 1524 K38Nal 308 409

TABLE 41 OSK1 Analogues with Improved Selectivity at Kv1.3 over Kv1.1 (whole cell patch clamp ePhys). Kv1.3 Selectivity SEQ Kv1.3 Kv1.1 (=Kv1.1/Kv.3 ID NO: Analogue (IC50, pM) (IC50, pM) IC50) 25 wild-type 39  202 5 1441 S11R 40  9130 228 1502 S11Nal 1490 85324 57 1410 R12A 25  440 17 1474 L15E 190 65014 342 1423 K27A 289  10085* 35 1424 M29A 33  3472 105 1454 M29R 760 23028 30 1425 N30A 766 10168 14 1428 K34A 16 12754 797 1521 H34Nal 215 29178 136 1489 H34E 1322 39352 30 1490 T36E 1921 83914 44 1491 P37E 241 15699 65 *PatchXpress data

TABLE 42 OSK1 Analogues with Multiple Amino Acid Substitutions. SEQ # Amino WB/IL-2 WB/IFNg ID Acid Amino Acid (IC50, (IC50, NO: Changes Changes nM) nM) 4988 2 M29A, H34Nal 0.087 0.102 296 2 E16K, K20D 1.579 1.32 4986 2 I4K(Gly), H34A 0.470 0.451 4987 2 H34A, K38K(Gly) 1.249 2.380 4985 2 K19K(Gly), H34A 1.514 1.633 1392 3 R12A, E16K, K20D 0.041 0.092 4990 3 E16K, K20D, H34A 65.462 26.629 298 3 E16K, K20D, T36Y 0.639 0.923 4991 4 S11Nal, R12A, M29A, 12.665 14.151 H34Nal 1396 5 E16K, K20D, 3.941 6.988 des36-38 1395 5 GGGGS-Osk1 1.357 2.204 1274 5 E16K, K20D, T36G, 2.636 3.639 P37G, K38G 4992 5 S11Nal, R12A, M29A, 3.511 5.728 H34Nal, P37E 4994 5 S11Nal, R12A, M23F, 8.136 22.727 M29A H34Nal 1398 5 R12P, E16K, K20D, 0.527 0.736 T37Y, K38Ne 1397 5 R12P, E16Om, K20E, 6.611 18.454 T37Y, K38Ne 4995 6 S11Nal, R12A, M23Ne, 14.32 68.158 M29A, H34Nal, P37E 4916 7 des1, V2G, R12A, E16K, 1.499 2.244 K19R, K20D, H34A 4993 7 S11Nal, R12A, L15E, >100 >100 M29A H34Nal, T36E, P37E 4989 12 des1-7, E16K, K20D, 8.179 8.341 des36-38

TABLE 43 Bioactivity of OSK1 and OSK1 peptide analog conjugates with half-life-extending moieties as indicated. F¹ (and F² if WB/IL-2 WB/IFNg present) Short-hand Designation (IC50, nM) (IC50, nM) PEG 20k PEG-OSK1[Ala12] 0.270 0.137 PEG 20k PEG-OSK1[Ala29] 5.756 5.577 PEG 20k PEG-OSK1[Ala29, 1Nal34] 0.049 0.081 PEG 20k PEG-OSK1[1Nal11] 0.019 0.027 PEG 20k PEG-ShK 0.046 0.065 FcLoop FcLoop-G2-OSK-G2 0.028 0.056 FcLoop FcLoop-G4-OSK-G2 0.150 0.195 FcLoop FcLoop-G2-ShK-G2 0.109 0.119 PEG-CH2 20k PEG-L2-6H-L3-CH2- 8.325 50.144 L10-OsK1(1-38) CH2 L2-6H-L3-CH2-L10- 38.491 55.162 OsK1(1-38) Fcloop structures G2-OSK1-G2 (SEQ ID N0: 976), G4-OSK1-G2 (SEQ ID NO: 979), and G2-SHK-G2 (SEQ ID N0: 977) are described in Example 49, and CH2-L10-OSK1(1-38) SEQ ID NO: 4917 is described in Example 54.

Example 56 Design and Expression of Monovalent Fc-Fusion Molecules

There may be pharmacokinetic or other reasons, in some cases, to prefer a monovalent dimeric Fc-toxin peptide fusion (as represented schematically in FIG. 2B) to a (“bivalent”) dimer (as represented schematically in FIG. 2C). However, conventional Fc fusion constructs typically result in a mixture containing predominantly dimeric molecules, both monovalent and bivalent. Monovalent dimeric Fc-toxin peptide fusions (or “peptibodies”), including monovalent dimeric Fc-OSK1 peptide analog fusions and Fc-ShK peptide analog fusions, can be isolated from conditioned media which also contains bivalent dimeric Fc-toxin peptide, and dimeric Fc lacking the toxin peptide fusion. Separation of all three species can be accomplished using ion exchange chromatography, for example, as described in Examples 1, 2, and 41 herein.

A number of other exemplary ways that a monovalent dimeric Fc-toxin peptide fusion can be produced with greater efficiency are provided here, including for the production of monovalent dimeric Fc-OSK1 peptide analog fusions:

(1) Co-expressing equal amounts of Fc and Fc-toxin peptide in the same cells (e.g. mammalian cells). With the appropriate design, a mixture of bivalent dimeric Fc-toxin peptide fusion, monovalent dimeric Fc-toxin peptide fusion and dimeric Fc will be produced and released into the conditioned media. The monovalent dimeric Fc-toxin peptide can be purified from the mixture using conventional purification methods, for example, methods described in Examples 1, 2, and 41 herein.

(2) Engineering and recombinantly expressing in mammalian cells a single polypeptide construct represented by the following schematic: Signal peptide-Fc-furin cleavage site-linker-furin cleavage site-Fc-toxin peptide Furin cleavage occurs as the molecule travels through the endoplasmic reticulum and the intra-molecular Fc pairing (resulting in monovalent dimeric Fc-toxin peptide fusion) can occur preferentially to intermolecular Fc pairing (resulting in dimeric Fc-toxin peptide being expressed into conditioned medium; FIG. 87A-B).

By way of example of method (2) above, a DNA construct was produced for recombinant expression in mammalian cells of the following schematic polypeptide construct: Signal peptide-Fc-furin cleavage site-linker-furin cleavage site-Fc-ShK(2-35)

The DNA construct had the following nucleotide coding sequence:

atggaatggagctgggtctttctcttcttcctg SEQ ID NO: 5007 tcagtaacgactggtgtccactccgacaaa actcacacatgcccaccgtgcc cagcacctgaactcctggggggaccgtca gtcttcctcttccccccaaaacccaaggac accctcatgatctcccggacccc tgaggtcacatgcgtggtggtggacgtga gccacgaagaccctgaggtcaagttca actggtacgtggacggcgtggaggt gcataatgccaagacaaagccgcggga ggagcagtacaacagcacgtaccgtgt ggtcagcgtcctcaccgtcctgcac caggactggctgaatggcaaggagtacaa gtgcaaggtctccaacaaagccctccca gcccccatcgagaaaaccatct ccaaagccaaagggcagccccgagaac cacaggtgtacaccctgcccccatcccgg gatgagctgaccaagaaccag gtcagcctgacctgcctggtcaaaggcttcta tcccagcgacatcgccgtggagtgggaga gcaatgggcagccggagaa caactacaagaccacgcctcccgtgctgga ctccgacggctccttcttcctctacagcaa gctcaccgtggacaagagcag gtggcagcaggggaacgtcttctcatgctc cgtgatgcatgaggctctgcacaaccac tacacgcagaagagcctctccctg tctccgggtaaacgaggcaagagggctg tggggggcggtgggagcggcggcggg ggctcaggtggcgggggaagtgg cgggggagggagtggagggggaggg agtggaggcgggggatccggcggggg gggtagcaagcgtcgcgagaagcg ggataagacccatacctgccccccctgt cccgcgcccgagttgctcgggggcccc agcgtgtttttgtttcctcccaagcctaa agatacattgatgattagtagaacaccc gaagtgacctgtgtcgtcgtcgatgtctc tcatgaggatcccgaagtgaaattcaa ttggtatgtcgatggggtcgaagtccaca acgctaaaaccaaacccagagaaga acagtataattctacctatagggtcgtg tctgtgttgacagtgctccatcaagattgg ctcaacgggaaagaatacaaatgtaa agtgagtaataaggctttgcccgctcct attgaaaagacaattagtaaggctaagg gccaacctagggagccccaagtctat acactccctcccagtagagacgagct gaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatcccagcga catcgccgtggagtgggagagcaatg ggcagccggagaacaactacaaga ccacgcctcccgtgctggactccgacg gctccttcttcctctacagcaagctcaccg tggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatg aggctctgcacaaccactacacgcag aagagcctctccctgtctccgggtaaag gaggaggaggatccggaggaggag gaagcagctgcatcgacaccatcccc aagagccgctgcaccgccttccagtgc aagcacagcatgaagtaccgcctgag cttctgccgcaagacctgcggcacctg ctaa//.

The resulting expressed polypeptide (from vector pTT5-Fc-Fc-L10-Shk(2-35)) had the following amino acid sequence before furin cleavage (the first 19 residues are a signal peptide sequence; furin cleavage sites are underlined):

mewswvflfflsvttgvhsdkthtcppcpape SEQ ID NO: 5008 llggpsvflfppkpkdtlmisrtpevtcvvv dvshedpevkfnwyvdgvevhna ktkpreeqynstyrvvsvltvlhqdwlngkey kckvsnkalpapiektiskakgqprepqvytlpp srdeltknqvsltclvkgfypsdiavewesngqp ennykttppvldsdgsfflyskltvdksrwqqgn vfscsvmhealhnhytqkslslspgkrgkravgg ggsggggsggggsggggsggggsggggsggg gskrrekrdkthtcppcpapellggpsvflfp pkpkdtlmisrtpevtcvvvdvshedpevkfnwy vdgvevhnaktkpreeqynstyrvvsvltvlhqd wlngkeykckvsnkalpapiektiskakgqpre pqvytlppsrdeltknqvsltclvkgfypsdia vewesngqpennykttppvldsdgsfflys kltvdksrwqqgnvfscsvmhealhnhy tqkslslspgkggggsggggsscidtipks rctafqckhsmkyrlsfcrktcgtc//.

FIG. 87A-B demonstrates recombinant expression of a monovalent dimeric Fc-L-ShK(2-35) molecule product expressed by and released into the conditioned media from transiently transfected mammalian cells. FIG. 88 shows results from a pharmacokinetic study on the monovalent dimeric Fc-ShK(1-35) in SD rats. Serum samples were added to microtiter plates coated with an anti-human Fc antibody to enable affinity capture. Plates were then washed, captured samples were released by SDS and run on a polyacrylamide gel. Samples were then visualized by western blot using an anti-human Fc-specific antibody and secondary-HRP conjugate. The MW of bands from serum samples is roughly identical to the original purified material, suggesting little, if any, degradation of the protein occurred in vivo over a pro-longed half-life, in spite of the presence of Arg at position 1 of the ShK(1-35) sequence.

(3) Similar to (2) above, a Fc-toxin peptide fusion monomer can be conjugated with an immunoglobulin light chain and heavy chain resulting in a monovalent chimeric immunoglobulin-Fc-toxin peptide molecule. We have termed an immunoglobulin (light chain+heavy chain)-Fc construct a “hemibody”; such “hemibodies” containing a dimeric Fc portion can provide the long half-life typical of a dimeric antibody. The schematic representation in FIG. 92A-C illustrates an embodiment of a hemibody-toxin peptide fusion protein and its recombinant expression by mammalian cells.

If the antibody chosen is a target specific antibody (e.g., an anti-Kv1.3 or anti-IKCa1 antibody), the chimeric molecule may also enhance the targeting efficiency of the toxin peptide. FIG. 91A-B demonstrates that such chimeric molecules, in this example Fc-L10-ShK(2-35) dimerized with human IgG1 or human IgG2 light and heavy chains, can be expressed and released into the conditioned media from transfected mammalian cells.

Example 57 Osk1 PEGylated at Residue 4 by Oxime Formation

[Dpr^((AOA)-PEG))4]Osk1 Peptide Synthesis of reduced [Dpr^((AOA))4]Osk1. [Dpr^((AOA))4]Osk1, having the sequence:

(SEQ ID NO: 5009) GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK can be synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Fmoc-Lys(Boc)-Wang resin (Novabiochem). N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids can be purchased from Novabiochem. The following side-chain protection strategy can be employed: Asp(O^(t)Bu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(N^(ε)-Boc), Ser(O^(t)Bu), Thr(O^(t)Bu) and Tyr(O^(t)Bu). Dpr(AOA), i.e., N-α-Fmoc-N-b-(N-t.-Boc-amino-oxyacetyl)-L-diaminopropionic acid, can be purchased from Novabiochem (Cat. No. 04-12-1185). The protected amino acid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions can be carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes then 15 minutes. Following synthesis and removal of the N-terminal Fmoc group, the resin can be then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin can be deprotected and released from the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O (90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. The volatiles can be then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The [Dpr^((AOA))4]Osk1 peptide can be then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phase high-performance liquid chromatography can be performed on C18, 5 μm, 2.2 cm×25 cm) column. The [Dpr^((AOA))4]Osk1 peptide is dissolved in 50% aqueous acetronitrile containing acetic acid and amionooxyacetic acid and loaded onto a preparative HPLC column. Chromatographic separations can be achieved using linear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA), typically 5-95% over 90 minutes at 15 mL/min. Preparative HPLC fractions can be characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized.

Osk1 peptide analog PEGylated at residue 4 by oxime formation: [Dpr^((AOA)-PEG))4]Osk1 (i.e., GVI[Dpr^((AOA-PEG))]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK//SEQ ID NO:5010) can be made as follows. Lyophilized [Dpr^((AOA))4]Osk1 peptide can be dissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molar excess of MeO-PEG-aldehyde, CH₃O—[CH₂CH₂O]_(n)—CH₂CH₂CHO (average molecular weight 20 kDa). The aminoxyacetyl group within the peptide at residue 4 reacts with the aldehyde group of the PEG to form a covalent oxime linkage. The reaction can be left for 24 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono PEGylated [Dpr^((AOA)-PEG))4]Osk1 peptide can be then isolated using a HiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product, mono-PEGylated [Dpr^((AOA)-PEG))4]Osk1 peptide, can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.

Folding of [Dpr^((AOA)-PEG))4]Osk1 (Disulphide bond formation). The mono-PEGylated [Dpr^((AOA)-PEG))4]Osk1 peptide can be dissolved in 20% AcOH in water (v/v) and can be then diluted with water to a concentration of approximately 0.15 mg peptide mL, the pH adjusted to about 8.0 using NH₄OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process can be monitored by LC-MS analysis. Following this, folded mono-PEGylated [Dpr^((AOA)-PEG))4]Osk1 can be purified using by reversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated) [Dpr^((AOA)-PEG))4]Osk1 peptide disulfide connectivity can be C1-C4, C2-C5, and C3-C6.

ABBREVIATIONS

Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific circumstances.

Ac acetyl (used to refer to acetylated residues) AcBpa acetylated p-benzoyl-L-phenylalanine ADCC antibody-dependent cellular cytotoxicity Aib aminoisobutyric acid bA beta-alanine Bpa p-benzoyl-L-phenylalanine BrAc bromoacetyl (BrCH₂C(O) BSA Bovine serum albumin Bzl Benzyl Cap Caproic acid COPD Chronic obstructive pulmonary disease CTL Cytotoxic T lymphocytes DCC Dicylcohexylcarbodiimide Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl ESI-MS Electron spray ionization mass spectrometry Fmoc fluorenylmethoxycarbonyl HOBt 1-Hydroxybenzotriazole HPLC high performance liquid chromatography HSL homoserine lactone IB inclusion bodies KCa calcium-activated potassium channel (including IKCa, BKCa, SKCa) Kv voltage-gated potassium channel Lau Lauric acid LPS lipopolysaccharide LYMPH lymphocytes MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry Me methyl MeO methoxy MHC major histocompatibility complex MMP matrix metalloproteinase 1-Nap 1-napthylalanine NEUT neutrophils Nle norleucine NMP N-methyl-2-pyrrolidinone PAGE polyacrylamide gel electrophoresis PBMC peripheral blood mononuclear cell PBS Phosphate-buffered saline Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl PCR polymerase chain reaction Pec pipecolic acid PEG Poly(ethylene glycol) pGlu pyroglutamic acid Pic picolinic acid pY phosphotyrosine RBS ribosome binding site RT room temperature (25° C.) Sar sarcosine SDS sodium dodecyl sulfate STK serine-threonine kinases t-Boc tert-Butoxycarbonyl tBu tert-Butyl THF thymic humoral factor Trt trityl 

1. A method of mitigating a relapse of a symptom of multiple sclerosis, comprising administering to a patient, who has been diagnosed with multiple sclerosis, an effective amount of a composition of matter of the formula (X¹)_(a)—(F¹)_(d)—(X²)_(b)—(F²)_(e)—(X³)_(c) and multimers thereof, wherein: F¹ and F² are half-life extending moieties, wherein F¹ or F², or both is a polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunonlobulin light chain and heavy chain an immunoglobulin F_(c) domain or portion thereof a CH2 domain of F_(c), an F_(c) domain loop, an albumin, an albumin-binding protein, a transthyretin, a thyroxine-binding globulin, or a ligand that has an affinity for a long half-life serum protein, said ligand being selected from the group consisting of peptide ligands and small molecule ligands; and d and e are each independently 0 or 1, provided that at least one of d and e is 1; X¹, X², and X³ are each independently -(L)_(f)-P-(L)_(g)-, and f and g are each independently 0 or 1; P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, and at least one P is an OSK1 peptide analog comprising the amino acid sequence of SEQ ID NO: 4916; L is a linker; and a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1, such that the symptom of multiple sclerosis is mitigated.
 2. A method of treating an autoimmune disorder, comprising administering to a patient who has been diagnosed with an autoimmune disorder selected from multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease (IBD), contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus, a therapeutically effective amount of a composition of matter of the formula (X¹)_(a)—(F¹)_(d)—(X²)_(b)—(F²)_(e)—(X³)_(c) and multimers thereof, wherein: F¹ and F² are half-life extending moieties, wherein F¹ or F², or both is a polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin light chain and heavy chain an immunoglobulin F_(c) domain or portion thereof a CH2 domain of F_(c) an F_(c) domain loop an albumin an albumin-binding protein, a transthyretin, a thyroxine-binding globulin, or a ligand that has an affinity for a long half-life serum protein, said ligand being selected from the group consisting of peptide ligands and small molecule ligands; and d and e are each independently 0 or 1, provided that at least one of d and e is 1; X¹, X², and X³ are each independently -(L)_(f)-P-(L)_(g)-, and f and g are each independently 0 or 1; P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, and at least one P is an OSK1 peptide analog comprising the amino acid sequence of SEQ ID NO: 4916; L is a linker; and a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1, whereby at least one symptom of the disorder is alleviated in the patient.
 3. The method of claim 1 or claim 2, wherein F¹ or F², or both, of the composition of matter comprises a human IgG Fc domain or a portion thereof.
 4. The method of claim 1 or claim 2, wherein F¹ and F² of the composition of matter are different half-life extending moieties.
 5. The method of claim 1 or claim 2, wherein F¹ or F², or both, of the composition of matter comprises a sequence selected from SEQ ID NOS: 2, 4, 70, 71, 72, 74, 75, 76, 1340 through 1342, and 1359 through 1363 as set forth in FIGS. 3, 4, 11A-C, 12A-C, and 12E-F.
 6. The method of claim 1 or claim 2, wherein F¹ or F², or both, the composition of matter comprises a biologically suitable polymer or copolymer.
 7. The method of claim 1 or claim 2, in which the toxin peptide of the composition of matter is inserted into a human IgG1 Fc domain loop.
 8. The method of claim 1 or claim 2, wherein the C-terminal carboxylic acid moiety of the OSK1 peptide analog of the composition of matter is replaced with a moiety selected from (A) —COOR, where R is independently (C₁-C₈)alkyl, haloalkyl, aryl or heteroaryl; (B) —C(═O)NRR, where R is independently hydrogen, (C₁-C₈)alkyl, haloalkyl, aryl or heteroaryl; and (C) —CH₂OR where R is hydrogen, (C₁-C₈) alkyl, aryl or heteroaryl.
 9. The method of claim 1 or claim 2, wherein the OSK1 peptide analog of the composition of matter is conjugated to an acyl, aryl, fatty acid, or polyethylene glycol (PEG) via: (a) 1, 2, 3 or 4 amino functionalized sites in the OSK1 peptide analog; (b) 1, 2, 3 or 4 thiol functionalized sites in the OSK1 peptide analog; (c) 1 or 2 ketone functionalized sites in the OSK1 peptide analog; (d) 1 or 2 azido functionalized sites in the OSK1 peptide analog; (e) 1 or 2 carboxyl functionalized sites in the OSK1 peptide analog; (f) 1 or 2 aminooxy functionalized sites in the OSK1 peptide analog; or (g) 1 or 2 seleno functionalized sites in the OSK1 peptide analog.
 10. The method of claim 1 or claim 2, wherein the OSK1 peptide analog of the composition of matter is conjugated to a polyethylene glycol (PEG) via: (a) 1, 2, 3 or 4 amino functionalized sites of the PEG; (b) 1, 2, 3 or 4 thiol functionalized sites of the PEG; (c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG; (d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG; (e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG; or (f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of the PEG.
 11. The method of claim 1 or claim 2, wherein the OSK1 peptide analog of the composition of matter is covalently linked at its N-terminal to a moiety selected from acyl, aryl, fatty acid, or polyethylene glycol.
 12. The method of claim 1 or claim 2, wherein the OSK1 peptide analog of the composition of matter is covalently linked at its N-terminal to a polyethylene glycol. 